Anti-cd4 antibody

ABSTRACT

An anti-CD4 antibody which binds to CD4, has a high affinity and has a high effector activity, such as an antibody-dependent cellular cytotoxicity (ADCC activity) or complement-dependent cellular cytotoxicity (CDC activity), is required for a disease relating to a CD4-expressing cell. 
     The present invention can provide a monoclonal antibody or an antibody fragment thereof, which binds to a CD4 extracellular region with high affinity and also exhibits a high ADCC activity or a high CDC activity; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which contains the DNA; a transformant obtainable by introducing the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a therapeutic agent using the antibody or the antibody fragment thereof or a diagnostic agent using the antibody or the antibody fragment thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a monoclonal antibody or an antibodyfragment thereof, which binds to human CD4 extracellular region with ahigh affinity and also exhibits a high antibody-dependent cellularcytotoxicity (hereinafter, referred to as “ADCC activity”) or a highcomplement-dependent cellular cytotoxicity (hereinafter, referred to as“CDC activity”); a hybridoma which produces the antibody; a DNA whichencodes the antibody; a vector which contains the DNA; a transformantobtainable by introducing the vector; a process for producing anantibody or an antibody fragment thereof using the hybridoma or thetransformant; and a therapeutic agent and a diagnostic agent using theantibody or the antibody fragment thereof.

2. Brief Description of the Background Art

Cluster of differentiation 4 (hereinafter, referred to as “CD4”) is aglycoprotein having a molecular weight of about 55 kDa, which isexpressed on the cell surface of most of thymic cells, about ⅔ ofperipheral blood T cells, monocytes, and macrophage. CD4 is a type Itransmembrane protein in which four immunoglobulin superfamily domains(designated in order as D1 to D4 from the N terminal to the cellmembrane side) are present on the outside of the cells, and two N-linkedsugar chains in total are bound to the domains D3 to D4. CD4 binds to amajor histocompatibility complex (MHC) class II molecule through D1 andD2 domains, and then activates the T cells. Further, it is also knownthat CD4 polymerizes through D3 and D4 domains. D1 domain of CD4 isknown to serve as a receptor for a human immunodeficiency virus(hereinafter, referred to as “HIV”) (Non-Patent Literature 1).

CD4 is also known as T4, and the gene has been cloned in 1985(Non-Patent Literature 2), and the DNA sequence, the amino acid sequenceand the three-dimensional structure of CD4 are publicly available from aknown database. For example, these can be obtained by reference toAccession Nos. P01730 (SWISSPROT), M12807 (EMBL), and the like.

The anti-CD4 monoclonal antibody OKT4 was first confirmed as amonoclonal antibody (hereinafter, referred to as “mAb”) which binds toCD4 (Non-Patent Literature 3). Since then, a large number of monoclonalantibodies against CD4 (hereinafter, referred to as “anti-CD4 mAbs”)have been reported. Most of anti-CD4 mAbs reported hitherto are known torecognize D1 domain (Non-Patent Literature 1). Some of anti-CD4 mAbs areunder clinical development for the purpose of treating cancers, immunediseases, and infections. For example, based on the fact that thebinding between CD4 and HIV is essential for the infection of HIV, anantibody which recognizes D1 domain of CD4 can inhibit the infection ofHIV, under the development as an HIV therapeutic agent.

Examples of the anti-CD4 mAb developed as a therapeutic agent forcancers or immune diseases include zanolimumab (6G5) (Non-PatentLiterature 4), keliximab (CE9.1) (Non-Patent Literature 5), and thelike. These antibodies are antibodies which exert their medicinalefficacy by specifically attacking CD4-expressing cells which are targetcells, and it is considered that the mechanism of medicinal efficacy ismainly due to an ADCC activity (Non-Patent Literatures 6 and 7).Meanwhile, these antibodies are shown to be devoid of a CDC activitywhich is generally known as one of the main mechanism of medicinalefficacy of therapeutic antibodies, like ADCC (Non-Patent Literatures 6and 7).

The potency of CDC activity is different depending on the subclass ofantibodies, and human IgG1 and IgG3 subclasses have a high CDC activity.It is known that the intensity of CDC activity among the subclasses isgenerally in the order of IgG3>IgG1<<IgG2≈IgG4. In addition, there is acase where the CDC activity of an antibody is exerted or is not exerteddepending on an antigen to which the antibody binds (Non-PatentLiterature 11). Accordingly, every antibody cannot always exhibit theCDC activity.

Among the anti-CD4 mAbs confirmed hitherto, little is known of theantibodies having a CDC activity. Uniquely, even though it is known thatOKT4 exhibits a CDC activity against a CD4-positive human cell line onlywhen a rabbit serum which is high in complement number is used(Non-Patent Literature 8), no examples of an antibody which exhibits aCDC activity against a CD4-positive human cell line when a humancomplement is used have been reported.

A dissociation constant K_(D) of an antibody for several known anti-CD4mAbs has been reported. For example, antibodies shown in the followingtable have been reported to have a dissociation constant K_(D) of about7 to 0.01 nM (Table 1). Upon the calculation of dissociation constants,there is a possibility that measurement values vary depending on assayequipments, assay methods, and analysis methods. Therefore, whencomparing dissociation constants, the values which were measured andanalyzed under the same conditions are required.

TABLE 1 Clone Name Dissociation Constant [M] 6G5* 7.1 × 10⁻⁹  CE9.1* 3.2× 10⁻¹¹ Leu-3a** 1.0 × 10⁻¹¹ 13B8.2*** 5.0 × 10⁻⁹  *WO97/13852,**Non-Patent Literature 9, ***Non-Patent Literature 10

-   Non-Patent Literature 1: Leucocyte Typing VI, 49 (1997)-   Non-Patent Literature 2: Cell, 42, 93 (1985)-   Non-Patent Literature 3: Proc. Natl. Acad. Sci. USA, 76, 4061 (1979)-   Non-Patent Literature 4: Blood, 109, 4655 (2007)-   Non-Patent Literature 5: Lancet, 352, 1109 (1998)-   Non-Patent Literature 6: Clin Immunol Immunopathol, 84, 73 (1997)-   Non-Patent Literature 7: Cancer Res, 67, 9945 (2007)-   Non-Patent Literature 8: J Immunol, 164, 1925 (2000)-   Non-Patent Literature 9: J Immunol, 145, 2839 (1990)-   Non-Patent Literature 10: Clinical Immunology, 119, 38 (2006)-   Non-Patent Literature 11: J. Immunol., 174, 5706 (2005)

SUMMARY OF THE INVENTION

An object of the present invention is to provide a monoclonal antibodyor an antibody fragment thereof, which binds to a CD4 extracellularregion with high affinity and also exhibits a high ADCC activity or CDCactivity; a hybridoma which produces the antibody; a DNA which encodesthe antibody; a vector which contains the DNA; a transformant obtainableby introducing the vector; a process for producing an antibody or anantibody fragment thereof using the hybridoma or the transformant; and atherapeutic agent or a diagnostic agent using the antibody or theantibody fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a construction process of a CD4 expression vector.

FIG. 2 shows a construction process of a CD4-Fc expression vector.

FIGS. 3(A) to 3(D) show the reactivity of an anti-CD4 rat monoclonalantibody and an anti-CD4 mouse antibody OKT4 for the human T celllymphoma cell line HPB-ALL in the FCM analysis. The graph is dividedinto (A) to (D) according to the subclass of antibodies. The abscissarepresents a concentration of each of the antibodies, and the ordinaterepresents the mean fluorescence intensity, MFI value. In FIG. 3(A), represents an anti-CD4 rat monoclonal antibody KM4065, and x representsan anti-CD4 rat monoclonal antibody KM4069. In FIG. 3(B), x representsan anti-CD4 rat monoclonal antibody KM4068. In FIG. 3(C), ▪ representsan anti-CD4 rat monoclonal antibody KM4066, and □ represents an anti-CD4rat monoclonal antibody KM4067. In FIG. 3(D),  represents an anti-CD4mouse monoclonal antibody OKT4.

FIG. 4 shows a complement-dependent cellular cytotoxicity (CDC activity)of an anti-CD4 rat monoclonal antibody and an anti-CD4 mouse monoclonalantibody OKT4 on a CD4-expressing transfectant. The ordinate representsa cellular cytotoxicity rate (%), and the abscissa represents anantibody concentration of an anti-CD4 monoclonal antibody.  representsan anti-CD4 rat monoclonal antibody KM4066, ▪ represents an anti-CD4 ratmonoclonal antibody KM4067, - represents an anti-CD4 rat monoclonalantibody KM4068, and ♦ represents an anti-CD4 mouse monoclonal antibodyOKT4.

FIG. 5 shows a cloning process of an anti-CD4 rat monoclonal antibody.

FIG. 6 shows a construction process of an anti-CD4 chimeric antibodyexpression vector.

FIG. 7 shows a construction process of expression vectors of anti-CD4human antibodies 6G5-1 and 6G5-113F.

FIG. 8 shows the reactivity of various anti-CD4 antibodies in thebinding ELISA. The abscissa represents a concentration of each of theantibodies, and the ordinate represents a binding activity of each ofthe antibodies. The reactivity of each antibody against recombinanthuman CD4 is shown. Δ in the broken line represents a monoclonalantibody 6G5-1, Δ in the solid line represents an anti-CD4 humanantibody 6G5-P, ▴ represents an anti-CD4 human antibody 6G5-113F, represents an anti-CD4 chimeric antibody KM4045, ▪ represents ananti-CD4 chimeric antibody KM4046, □ represents an anti-CD4 chimericantibody KM4047, x in the broken line represents an anti-CD4 chimericantibody KM4048, and x in the solid line represents an anti-CD4 chimericantibody KM4049.

FIGS. 9(A) to 9(D) show an antibody-dependent cellular cytotoxicity(ADCC activity) of various anti-CD4 antibodies on various human T celllines. The ordinate represents a cellular cytotoxicity rate (%), and theabscissa represents an antibody concentration of each antibody. Δ in thebroken line represents a monoclonal antibody 6G5-1, Δ in the solid linerepresents a monoclonal antibody 6G5-P, ▴ represents a chimeric antibody6G5-113F,  represents a chimeric antibody KM4045, ▪ represents achimeric antibody KM4046, □ represents a chimeric antibody KM4047, x inthe broken line represents a chimeric antibody KM4048, and x in thesolid line represents a chimeric antibody KM4049. FIG. 9(A) shows anADCC activity when HPB-ALL was used as a target cell. FIG. 9(B) shows anADCC activity when HUT78 was used as a target cell. FIG. 9(C) shows anADCC activity when SUP-T1 is used as a target cell. FIG. 9(D) shows anADCC activity when HPB-ALL was used as a target cell.

FIGS. 10(A) to 10(B) show a complement-dependent cellular cytotoxicity(CDC activity) of various anti-CD4 antibodies on a CD4-expressingtransfectant or a human lymphoma cell line. The ordinate represents acellular cytotoxicity rate (%), and the abscissa represents an antibodyconcentration of each antibody. FIG. 10(A) shows a CDC activity on aCD4-expressing transfectant. FIG. 10(B) shows a CDC activity on thehuman lymphoma cell line HPB-ALL. ♦ in the broken line represents ananti-CD4 human antibody 6G5-1, ♦ in the solid line represents ananti-CD4 human antibody 6G5-113F, ▴ represents an anti-CD4 chimericantibody KM4045,  represents an anti-CD4 chimeric antibody KM4046, ▪represents an anti-CD4 chimeric antibody KM4047, - represents ananti-CD4 chimeric antibody KM4048, ▪ represents an anti-CD4 chimericantibody KM4049, and ▪ in the broken line represents an anti-CD4 mouseantibody OKT4.

FIG. 11 shows the reactivity of various anti-CD4 antibodies for aCD4-positive T cell lymphoma cell line HPB-ALLin the use of a flowcytometer. The abscissa represents an antibody concentration (μg/mL),and the ordinate represents a mean fluorescence intensity, MFI value. represents a chimeric antibody KM4045, □ represents a humanized antibodyHV0LV0, ◯ represents a humanized antibody HV2LV0, ⋄ represents ahumanized antibody HV3LV0, - represents a humanized antibody HV4LV0, xrepresents a humanized antibody HV4LV6, Δ in the broken line representsa human antibody 6G5-1, and Δ in the solid line represents a humanantibody 6G5-P.

FIGS. 12(A) to 12(C) show an antibody-dependent cellular cytotoxicity(ADCC activity) of various anti-CD4 antibodies on various human T celllymphoma lines. The ordinate represents a cellular cytotoxicity rate(%), and the abscissa represents an antibody concentration (ng/mL). represents a chimeric antibody KM4045, ◯ represents a humanized antibodyKM8045, ▴ in the broken line represents a human antibody 6G5-1, and ▴ inthe solid line represents a human antibody 6G5-P. FIG. 12(A) shows anADCC activity when HPB-ALL was used as a target cell. FIG. 12(B) showsan ADCC activity when HUT78 was used as a target cell. FIG. 12(C) showsan ADCC activity when CCRF-CEM was used as a target cell.

FIG. 13 shows anti-tumor effect (early stage cancer model) of eachantibody on the human T cell lymphoma cell line HH which had beentransplanted into a SCID mouse. The abscissa represents the number ofdays after tumor transplantation, and the ordinate represents a tumorvolume. x represents a negative control group, ▴ represents aKM8045-administered group, and  represents a 6G5-P-administered group.The bar represents a standard deviation.

FIGS. 14(A) and 14(B) show anti-tumor effects (metastasis model) ofindividual antibodies on CD4/EL4 cells which had been transplanted intoa C57BL/6 mouse. In FIG. 14(A), the ordinate represents a liver weightratio. In FIG. 14(B), the ordinate represents a kidney weight ratio. Thebar represents a standard deviation.

FIG. 15 shows anti-tumor effects (progressive cancer model) of eachantibody on an HH cell line which had been transplanted into a SCIDmouse. The abscissa represents the number of days after tumortransplantation, and the ordinate represents a tumor volume. xrepresents a negative control group, ▴ represents a group to which 20μg/head of KM8045 was administered,  represents a group to which 100μg/head of KM8045 was administered, and ▪ represents a group to which200 μg/head of KM8045 was administered. The bar represents a standarddeviation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the following (1) to (35):

(1) A monoclonal antibody against human CD4 or an antibody fragmentthereof, which has a dissociation constant (hereinafter, referred to as“K_(D)”) less than 1×10⁻⁹M of an antibody to an antigen, binds to anextracellular region of human CD4 with high affinity and has a highantibody-dependent cellular cytotoxicity (hereinafter, referred to as“ADCC activity”);(2) The monoclonal antibody or the antibody fragment thereof describedin the above (1), wherein the antibody is an antibody having a highcomplement-dependent cellular cytotoxicity (CDC activity);(3) The monoclonal antibody or the antibody fragment thereof describedin the above (2), wherein the antibody is an antibody which has a CDCactivity on a human cancer cell line expressing human CD4;(4) The monoclonal antibody or the antibody fragment thereof describedin any one of the above (1) to (3), wherein the monoclonal antibody is amonoclonal antibody which competes with an antibody in whichcomplementarity determining regions (hereinafter, referred to as “CDRs”)1 to 3 of a heavy chain (hereinafter, referred to as “H chain”) of theantibody comprise the amino acid sequences of SEQ ID NOs:51 to 53,respectively, and CDRs 1 to 3 of a light chain (hereinafter, referred toas “L chain”) of the antibody comprise the amino acid sequences of SEQID NOs:54 to 56, respectively, to bind to an extracellular region ofCD4;(5) The monoclonal antibody or the antibody fragment thereof describedin any one of the above (1) to (4), the monoclonal antibody is amonoclonal antibody which binds to the same epitope as an epitope in anextracellular region of CD4 to which an antibody in which CDRs 1 to 3 ofan H chain of the antibody comprise the amino acid sequences of SEQ IDNOs:51 to 53, respectively, and CDRs 1 to 3 of an L chain of theantibody comprise the amino acid sequences of SEQ ID NOs:54 to 56,respectively, binds;(6) The monoclonal antibody or the antibody fragment thereof describedin any one of the above (1) to (2), wherein the monoclonal antibody is amonoclonal antibody which competes with an antibody in which CDRs 1 to 3of an H chain of the antibody comprise the amino acid sequences of SEQID NOs:27 to 29, respectively, and CDRs 1 to 3 of an L chain of theantibody comprise the amino acid sequences of SEQ ID NOs:30 to 32,respectively, to bind to an extracellular region of CD4.

(7) The monoclonal antibody or the antibody fragment thereof describedin any one of the above (1) to (2) and (6), wherein the monoclonalantibody is a monoclonal antibody which binds to the same epitope in anextracellular region of CD4 as an epitope to which an antibody in whichCDRs 1 to 3 of an H chain of the antibody comprise the amino acidsequences of SEQ ID NOs:27 to 29, respectively, and CDRs 1 to 3 of an Lchain of the antibody comprise the amino acid sequences of SEQ ID NOs:30to 32, respectively, binds;

(8) The antibody or the antibody fragment thereof described in any oneof the above (1) to (7), wherein the monoclonal antibody is arecombinant antibody;(9) The recombinant antibody or the antibody fragment thereof describedin the above (8), wherein the recombinant antibody is a recombinantantibody selected from a human chimeric antibody, a humanized antibodyand a human antibody;(10) The recombinant antibody or the antibody fragment thereof describedin the above (9), wherein an H chain variable region (hereinafter,referred to as “VH”) of the antibody and an L chain variable region(hereinafter, referred to as “VL”) of the antibody comprise the aminoacid sequences of any one group selected from SEQ ID NO:16 and SEQ IDNO:26, respectively; and SEQ ID NO:12 and SEQ ID NO:22, respectively;(11) The recombinant antibody or the antibody fragment thereof describedin the above (9), wherein CDRs 1 to 3 of an H chain of the antibody andCDRs 1 to 3 of the L chain of an antibody comprise the amino acidsequences of any one group selected from SEQ ID NOs:51 to 53 and SEQ IDNOs:54 to 56, respectively; and SEQ ID NOs:27 to 29 and SEQ ID NOs:30 to32, respectively;(12) The humanized antibody or the antibody fragment thereof describedin the above (9), wherein the recombinant antibody is a humanizedantibody, and VH and VL of the antibody comprise the amino acidsequences of any one group selected from SEQ ID NO:77 and SEQ ID NO:78,respectively; SEQ ID NO:96 and SEQ ID NO:78, respectively; SEQ ID NO:98and SEQ ID NO:78, respectively; SEQ ID NO:100 and SEQ ID NO:78,respectively; and SEQ ID NO:100 and SEQ ID NO:102, respectively;(13) The antibody or the antibody fragment thereof described in any oneof the above (1) to (13), wherein the antibody fragment is an antibodyfragment selected from Fab, Fab′, F(ab′)₂, a single chain antibody(scFv), a dimerized V region (diabody), a disulfide stabilized V region(dsFv) and a peptide comprising CDR;(14) A DNA which encodes the antibody or the antibody fragment thereofdescribed in any one of the above (1) to (13);(15) A recombinant vector which comprises the DNA described in the above(14);(16) A transformant obtainable by introducing the recombinant vectordescribed in the above (15) into a host cell;(17) A process for producing the monoclonal antibody or the antibodyfragment thereof described in any one of the above (1) to (13),comprising culturing the transformant described in the above (16) in amedium to produce and accumulate the antibody or the antibody fragmentthereof described in any one of the above (1) to (14) in the culture,and collecting the antibody or the antibody fragment thereof from theculture;(18) A method for immunologically detecting or measuring human CD4,comprising using the antibody or the antibody fragment thereof describedin any one of the above (1) to (13);(19) An agent for detecting or measuring human CD4, comprising using theantibody or the antibody fragment thereof described in any one of theabove (1) to (13);(20) A diagnostic agent for a disease relating to human CD4, comprisingusing the antibody or the antibody fragment thereof described in any oneof the above (1) to (13);(21) The diagnostic agent described in the above (20), wherein thedisease relating to human CD4-positive cell is a cancer;(22) The diagnostic agent described in the above (20), wherein thedisease relating to a human CD4-positive cell is an allergic disease oran autoimmune disease;(23) A therapeutic agent for a relating to human CD4-positive cell,comprising the antibody or the antibody fragment thereof described inany one of the above (1) to (13) as an active ingredient;(24) The therapeutic agent described in the above (23), wherein thedisease relating to a human CD4-positive cell is a cancer;(25) The therapeutic agent described in the above (23), wherein thedisease relating to a human CD4-positive cell is an allergic disease oran autoimmune disease;(26) A method for diagnosing a disease relating to a human CD4-positivecell, comprising detecting or measuring a human CD4-positive cell;(27) A method for diagnosing a disease relating to a human CD4-positivecell, comprising detecting or measuring a human CD4;(28) The method for diagnosing described in the above (26) or (27),wherein the disease relating to a human CD4-positive cell is a cancer;(29) The method for diagnosing described in the above (26) or (27),wherein the disease relating to a human CD4-positive cell is an allergicdisease or an autoimmune disease;(30) Use of the antibody or the antibody fragment thereof described inany one of the above (1) to (13) for the manufacture of a diagnosticagent for a disease relating to a human CD4-positive cell;(31) The use of the antibody or the antibody fragment thereof describedin the above (30), wherein the disease relating to a human CD4-positivecell is a cancer;(32) The use of the antibody or the antibody fragment thereof describedin the above (30), wherein the disease relating to a human CD4-positivecell is an allergic disease or an autoimmune disease;(33) Use of the antibody or the antibody fragment thereof described inany one of the above (1) to (13) for the manufacture of a therapeuticagent for a disease relating to a human CD4-positive cell;(34) The use of the antibody or the antibody fragment thereof describedin the above (33), wherein the disease relating to a human CD4-positivecell is a cancer; and(35) The use of the antibody or the antibody fragment thereof describedin the above (33), wherein the disease relating to a human CD4-positivecell is an allergic disease or an autoimmune disease.

The present invention can provide a monoclonal antibody or an antibodyfragment thereof, which specifically recognizes an extracellular regionof human CD4 and binds to the extracellular region with a high affinity,and also exhibits a high ADCC activity or a high CDC activity; ahybridoma which produces the antibody; a DNA which encodes the antibody;a vector which comprises the DNA; a transformant obtainable byintroducing the vector; a process for producing the antibody or theantibody fragment thereof using the hybridoma or the transformant; and atherapeutic agent and a diagnostic agent using the antibody or theantibody fragment thereof.

The present invention relates to a monoclonal antibody or an antibodyfragment thereof, which binds with a high affinity to an extracellularregion of CD4 and accordingly has a high antibody-dependent cellularcytotoxicity (hereinafter, referred to as “ADCC activity”).

CD4 of the present invention includes a polypeptide comprising the aminoacid sequence represented by SEQ ID NO:1 or EMBL Accession No. M12807; apolypeptide comprising an amino acid sequence in which one or more aminoacid residue(s) is/are deleted, substituted or added in the amino acidsequence represented by SEQ ID NO:1 or EMBL Accession No. M12807, andhaving the activity of CD4; a polypeptide comprising an amino acidsequence having at least 60% homology, preferably at least 80% homology,more preferably at least 90% homology, and most preferably at least 95%homology, with the amino acid sequence represented by SEQ ID NO:1 orEMBL Accession No. M12807, and having the activity of CD4; and the like.

The polypeptide comprising an amino acid sequence in which one or moreamino acid residue(s) is/are deleted, substituted and/or added in theamino acid sequence represented by SEQ ID NO:1 or EMBL Accession No.M12807 can be obtained, for example, by introducing a site-specificmutation into DNA encoding a polypeptide comprising the amino acidsequence represented by SEQ ID NO:1 by site-specific mutagenesis[Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989), Current Protocols in Molecular Biology,John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982),Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985),Nucleic Acids Research, 13, 4431 (1985), or Proc. Natl. Acad. Sci. USA,82, 488 (1985)] or the like. The number of amino acid residues which aredeleted, substituted or added is not particularly limited, and thenumber is preferably, 1 to dozens, such as 1 to 20, and more preferably1 to several, such as 1 to 5.

As a gene encoding CD4, the nucleotide sequence represented by SEQ IDNO:2 or EMBL Accession No. M12807 may be exemplified. As the geneencoding CD4, the gene encoding CD4 of the present invention alsoincluded a gene containing a DNA comprising a nucleotide sequence havingdeletion(s), substitution(s) or addition(s) of one or more nucleotidesin the nucleotide sequence represented by SEQ ID NO:2 or EMBL AccessionNo. M12807 and also encoding a polypeptide having the function of CD4; agene containing a DNA consisting of a nucleotide sequence having atleast 60% or higher homology, preferably 80% or higher homology, andmore preferably 95% or higher homology, with the nucleotide sequencerepresented by SEQ ID NO:2 or EMBL Accession No. M12807, and alsoencoding a polypeptide having the function of CD4; a gene consisting ofa DNA which hybridizes with a DNA having the nucleotide sequencerepresented by SEQ ID NO:2 or EMBL Accession No. M12807 under stringentconditions and also containing a DNA that encodes a polypeptide havingthe function of CD4; and the like.

In the present invention, the DNA which hybridizes under stringentconditions refers to a DNA which is obtained by colony hybridization,plaque hybridization, Southern blot hybridization, DNA microarray or thelike using a DNA having the nucleotide sequence represented by SEQ IDNO:2 or EMBL Accession No. M12807 as a probe. A specific example of suchDNA is a hybridized colony- or plaque derived DNA which can beidentified by performing hybridization at 65° C. in the presence of 0.7to 1.0 mol/L sodium chloride using a filter or slide glass with the PCRproduct or oligo DNA having immobilized thereon, and then washing thefilter or slide glass at 65° C. with a 0.1 to 2-fold concentration SSCsolution (1-fold concentration SSC solution: 150 mmol/L sodium chlorideand 15 mmol/L sodium citrate). Hybridization can be carried outaccording to the methods [Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Lab. Press (1989), Current Protocols inMolecular Biology, John Wiley & Sons (1987-1997); DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford University(1995)] and the like. Specifically, the DNA capable of hybridizationincludes DNA having at least 60% or more homology, preferably 80% ormore homology, more preferably 90% or more homology, and most preferably95% or more homology to the nucleotide sequence represented by SEQ IDNO:2 or EMBL Accession No. M12807.

In the nucleotide sequence of the gene encoding a protein of aeukaryote, genetic polymorphism is often recognized. The CD4 gene usedin the present invention also includes a gene in which smallmodification is generated in the nucleotide sequence by suchpolymorphism as the gene used in the present invention.

The number of the homology in the present invention may be a numbercalculated by using a homology search program known by the skilledperson, unless otherwise indicated. Regarding the nucleotide sequence,the number may be calculated by using BLAST [J. Mol. Biol., 215, 403(1990)] with a default parameter or the like, and regarding the aminoacid sequence, the number may be calculated by using BLAST2 [NucleicAcids Res., 25, 3389 (1997); Genome Res., 7, 649 (1997) with a defaultparameter;http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] orthe like.

As the default parameter, G (cost to open gap) is 5 for the nucleotidesequence and 11 for the amino acid sequence; -E (cost to extend gap) is2 for the nucleotide sequence and 1 for the amino acid sequence; -q(penalty for nucleotide mismatch) is -3; -r (reward for nucleotidematch) is 1; -e (expect value) is 10; -W (wordsize) is 11 residues forthe nucleotide sequence and 3 residues for the amino acid sequence; -y[dropoff (X) for blast extensions in bits] is 20 for blastn and 7 for aprogram other than blastn; -X (X dropoff value for gapped alignment inbits) is 15; and -Z (final X dropoff value for gapped alignment in bits)is 50 for blastn and 25 for a program other than blastn(http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

The polypeptide comprising a partial sequence of the amino acid sequencerepresented by SEQ ID NO:1 or EMBL Accession No. M12807 can be preparedaccording to a method known by the skilled person. For example, it canbe prepared by deleting a part of DNA encoding the amino acid sequencerepresented by SEQ ID NO:2 and culturing a transformant into which anexpression vector containing the DNA is introduced. Also, based on thepolypeptide or DNA prepared by using the above method, a polypeptidecomprising an amino acid sequence in which one or more amino acid(s)is/are deleted, substituted or added in a partial sequence of the aminoacid sequence represented by SEQ ID NO:1 or EMBL Accession No. M12807can be prepared in the same manner as described above. In addition, thepolypeptide comprising a partial sequence of the amino acid sequencerepresented by SEQ ID NO:1 or EMBL Accession No. M12807; or apolypeptide comprising an amino acid sequence in which one or more aminoacid(s) is/are deleted, substituted or added in a partial sequence ofthe amino acid sequence represented by SEQ ID NO:1 or EMBL Accession No.M12807 can be produced by a chemical synthesis method such asfluorenylmethoxycarbonyl (Fmoc) method or t-butyloxycarbonyl (tBoc)method.

In the present invention, the extracellular region of CD4 includes, forexample, regions predicted from the amino acid sequence of thepolypeptide represented by SEQ ID NO:1 by using conventionally knowntransmembrane region deducing program SOSUI(http://bp.nuap.nagoya-u.ac.jp/SOSUI/SOSUI_submit.), TMHMM ver. 2(http://www.cbs.dtu.dk/services/TMHMM-2.0/), ExPASy Proteomics Server(http://Ca.expasy.org/) or the like.

Examples of the extracellular region of CD4 in the present inventioninclude regions corresponding to N-terminal to position 394 in theextracellular domain predicted by SOSUI. The extracellular regioncomprises immunoglobulin superfamily domains (D1 to D4) and every domainis comprised in the predicted extracellular domain.

The extracellular region of CD4 in the present invention may be anystructure, so long as it has a structure equivalent to a structure whichcan be taken in a native state by the extracellular region of CD4 havingthe amino acid sequence represented by SEQ ID NO:1 or EMBL Accession No.M12807. The term “structure which can be taken in a native state by theextracellular region of CD4” refers to a native three-dimensional(comformational) structure of CD4 which is expressed on a cell membrane.

The function of CD4 in the present invention means that CD4 induces theactivation or differentiation of T cells by acting as a cofactor of a Tcell receptor when CD4 binds to an MHC class II-antigen complex which isexpressed on an antigen-presenting cell, through the T cell receptorwhich is expressed on T cells. In addition, examples of the function ofCD4 also includes its participation in the infection of T cells with HIVby such a manner that CD4 expressed on a cell membrane of T cells bindsto gp120 which is one of envelope proteins of a human immunodeficiencyvirus (HIV).

Binding of the antibody or antibody fragment of the present invention tothe extracellular region of CD4 can be confirmed by a method in whichthe binding ability of a cell expressing a specified antigen and anantibody for the specific antigen can be examined, for example, by aradioimmunoassay using a solid phase sandwich method or the like, or aconventionally known immunological detecting method for a cellexpressing CD4 using an enzyme immuno assay (ELISA) method, preferably afluorescent cell staining method or the like. Examples include afluorescent antibody staining method using the FMAT8100HTS system(manufactured by Applied Biosystem), [Cancer Immunol. Immunother., 36,373 (1993)], a fluorescent cell staining method using a flow cytometry,a surface plasmon resonance using the Biacore system (manufactured by GEHealthcare) and the like. In addition, it can also be confirmed by acombination of conventionally known immunological detecting methods[Monoclonal Antibodies—Principles and Practice, Third edition, AcademicPress (1996), Antibodies—A Laboratory Manual, Cold Spring HarborLaboratory (1988), Monoclonal Antibody Experiment Manual, KodanshaScientific (1987)] and the like.

In the present invention, as a cell expressing CD4 includes any cellswhich so long as express CD4. Examples include a cell which exists inhuman body, a cell line which is established from naturally existingcell in human body, and a cell line which is produced by using generecombinant techniques.

The cell which is naturally present in the human body includes a cellexpressing CD4 in the body of a patient suffering from autoimmunedisease, an allergic patient and a cancer patient. Examples include acell expressing CD4 among tumor cells obtained by biopsy or the like.

The cell line established from the cell which is naturally present inthe human body includes a cell line expressing CD4 among the cell linesobtained by establishing the above-described cells expressing CD4obtained from a cancer patient, such as a human T cell lymphoma cellline HPB-ALL (DSMZ NO: ACC483) or HUT78 (ATCC No: TIB 16).

Specific examples of the cell obtained by using gene recombinationtechnique includes a cell expressing CD4, which is prepared byintroducing an expression vector comprising cDNA encoding CD4 into aninsect cell, an animal cell, etc., and the like.

Examples of the antibody of the present invention include a monoclonalantibody or an antibody fragment thereof against human CD4 which has adissociation constant (hereinafter, referred to as “K_(D)”) less than1×10⁻⁹M of the antibody to CD4, binds to an extracellular region ofhuman CD4 with high affinity and has high ADCC activity.

Examples of the antibody of the present invention include a monoclonalantibody or an antibody fragment thereof against human CD4 which has adissociation constant “K_(D)” of the antibody of 1×10⁻⁹M or less to theantigen and has a high ADCC activity and a high complement-dependentcellular cytotoxicity (CDC activity). Examples of the antibody of thepresent invention also include an antibody or an antibody fragment whichhas CDC activity on a human cancer cell line expressing human CD4.

The monoclonal antibody of the present invention includes an antibodyproduced by a hybridoma and a recombinant antibody produced by atransformant transformed with an expression vector containing a geneencoding the antibody.

The monoclonal antibody is an antibody secreted by a single cloneantibody-producing cell, and recognizes only one epitope (also calledantigen determinant) and has uniform amino acid sequence (primarystructure).

In the present invention, the monoclonal antibody has a structurecomprising a heterotetramer consisting of two H chains and two L chains.A H chain comprises a H chain variable region (hereinafter referred toas “VH”) and a H chain constant region (hereinafter referred to as“CH”); and a L chain comprises a L chain variable region (hereinafterreferred to as “VL”) and a L chain constant region (hereinafter referredto as “CL”). In addition, CH comprises four domains: CH1 domain, hingedomain, CH2 domain and CH3 domain. Furthermore, a domain consisting ofCH2 domain and CH3 domain together is defined as “Fc region”, “Fcdomain” or simply “Fc” of an antibody.

Examples of the epitope include a single amino acid sequence, athree-dimensional structure formed by an amino acid sequence, an aminoacid sequence having a sugar chain bound thereto, a three-dimensionalstructure formed by an amino acid sequence having a sugar chain boundthereto, and the like, which a monoclonal antibody recognizes and bindsto. Examples of the epitope of the monoclonal antibody of the presentinvention include preferably the extracellular region of CD4, and anepitope corresponding to position 1 to 394 of the amino acid sequencerepresented by SEQ ID NO:1, more preferably an epitope comprising D1 orD2 of the extracellular region of CD4, and an epitope comprising D3 orD4 of the extracellular region of CD4 near a cell membrane and the like.

In the present invention, an antibody which binds to CD4 with highaffinity is an antibody which has enough affinity for a therapeuticantibody, preferably an antibody which binds to CD4 with dissociationconstant K_(D) value less than 1×10⁻⁹M, preferably less than 7×10⁻¹⁰M,more preferably less than 2×10⁻¹⁰M in terms of affinity. Zanolimumab(6G5) with which a clinical trial has already started has affinity withdissociation constant K_(D) value of 7.1×10⁻⁹M and exhibited hightherapeutic effects. Therefore, the antibody of the present inventioncan exhibit high therapeutic effects.

Affinity is measured by kinetic analysis, and for example, can bemeasured by using a Biacore T100 (manufactured by GE HealthcareBio-Sciences), or the like.

In the present invention, the term “dissociation is slow” means that avalue of a dissociation rate constant kd of an antibody calculated byBiacore T100 has a smaller value. The smaller dissociation rate constantrepresents that an antibody does not easily dissociate from anantigen-expressing cell. By increasing an amount of an antibody bindingto the cell surface to thereby extend the effective time of theantibody, then a high medicinal efficacy can be expected. A dissociationrate constant kd is measured, for example, using a Biacore T100(manufactured by GE Healthcare Bio-Sciences), and can be calculated bysoftware attached to the apparatus, Biacore T100 evaluation software(manufactured by Biacore).

As used herein, the term “antibody having a high ADCC activity” refersto an antibody having a higher ADCC activity than anti-CD4 antibodies6G5 and CE9.1 which had been reported to have an ADCC activity, whenADCC activities of plural antibodies were simultaneously measured forCD4-expressing cells, using a known assay method [Cancer Immunol.Immunother., 36, 373 (1993)].

The term “ADCC activity” refers to an activity which leads to thecytotoxicity to a target cell by such a manner that an antibody bound toan antigen on the target cell binds to an Fc receptor of an immune cellthrough an Fc region of the antibody, consequently resulting in theactivation of the immune cell (natural killer cell, etc.).

An Fc receptor (hereinafter referred to as “FcR”) is a receptor whichbinds to Fc region of an antibody and leads to various effectoractivities by binding to the antibody. An FcR corresponds to a subclassof an antibody, and IgG, IgE, IgA and IgM specifically binds to FcγR,FcεR, FcαR and FcμR, respectively. Moreover, an FcγR has subtypes ofFcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) and these have isoformsof FcγRIA, FcγRIB, FcγRIC, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA andFcγRIIIB, respectively. The above different FcγRs exist on differentcells (Annu. Rev. Immunol., 9:457-492 (1991)). In human, FcγRIIIBspecifically expresses on a neutrophil and FcγRIIIA expresses on amonocyte, a Natural Killer cell (NK cell) and some part of T cell.Binding of an antibody through FcγRIIIA leads to an NK cell-dependentADCC activity.

In the present invention, the term “antibody having a high CDC activity”refers to an antibody having a higher CDC activity than conventionalanti-CD4 antibodies, when CDC activities of plural antibodies weresimultaneously measured for CD4-expressing cells, using a humancomplement and a known CDC assay method [Cancer Immunol. Immunother.,36, 373 (1993)]. More preferred is an antibody having a higher CDCactivity than conventional anti-CD4 antibodies, when a CDC activity ofthe antibody was measured in the same manner as above for aCD4-expressing cell which is naturally present in the human body orCD4-positive cancer cell line. More specifically, such an antibody isone exhibiting a higher CDC activity than a conventional anti-CD4antibody 6G5 or OKT4, in the same experimental system.

The term “CDC activity” refers to an activity which leads to thecytotoxicity to a target cell by such a manner that an antibody bound toan antigen on the target cell activates a series of cascades (complementactivation pathways) containing complement-related protein groups inblood. In addition, protein fragments generated by the activation of acomplement can induce the migration and activation of immune cells.

Examples of the antibody of the present invention include a monoclonalantibody or an antibody fragment which binds to either D1 or D2 of theextracellular region of CD4, or D3 or D4 of the extracellular region ofCD4 near a cell membrane.

Further, among the antibodies of the present invention, examples of anantibody which recognizes D1 or D2 domain include a monoclonal antibodyin which a heavy chain constant region (hereinafter referred to as VH)of the antibody comprises the amino acid sequences of CDRs 1 to 3represented by SEQ ID NOs:33 to 35, respectively, and a light chainconstant region (hereinafter referred to as VL) of the antibodycomprises the amino acid sequences of CDRs 1 to 3 represented by SEQ IDNOs:36 to 38, respectively, and the like. In addition, among specificantibodies of the present invention, examples of an antibody whichrecognizes D3 or D4 domain include a monoclonal antibody in which VH ofthe antibody comprises the amino acid sequences of CDRs 1 to 3represented by SEQ ID NOs:51 to 53, respectively, and VL of the antibodycomprises the amino acid sequences of CDRs 1 to 3 represented by SEQ IDNOs:54 to 56, respectively; a monoclonal antibody in which VH of theantibody comprises the amino acid sequences of CDRs 1 to 3 representedby SEQ ID NOs:27 to 29, respectively, and VL of the antibody comprisesthe amino acid sequences of CDRs 1 to 3 represented by SEQ ID NOs:30 to32, respectively; a monoclonal antibody in which VH of the antibodycomprises the amino acid sequences of CDRs 1 to 3 represented by SEQ IDNOs:39 to 41, respectively, and VL of the antibody comprises the aminoacid sequences of CDRs 1 to 3 represented by SEQ ID NOs:42 to 44,respectively; and a monoclonal antibody in which VH of the antibodycomprises the amino acid sequences of CDRs 1 to 3 represented by SEQ IDNOs:45 to 47, respectively, and VL of the antibody comprises the aminoacid sequences of CDRs 1 to 3 represented by SEQ ID NOs:48 to 50,respectively.

Moreover, specific examples of the monoclonal antibody of the presentinvention include a monoclonal antibody wherein VH of the antibodycomprises the amino acid sequence represented by SEQ ID NO:16 and VL ofthe antibody comprises the amino acid sequence represented by SEQ IDNO:26; a monoclonal antibody wherein VH of the antibody comprises theamino acid sequence represented by SEQ ID NO:12 and VL of the antibodycomprises the amino acid sequence represented by SEQ ID NO:22; amonoclonal antibody wherein VH of the antibody comprises the amino acidsequence represented by SEQ ID NO:13 and VL of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:23; a monoclonalantibody wherein VH of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:14 and VL of the antibody comprises the aminoacid sequence represented by SEQ ID NO:24; a monoclonal antibody whereinVH of the antibody comprises the amino acid sequence represented by SEQID NO:15 and VL of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:25; and the like.

In addition, examples of the monoclonal antibody of the presentinvention include a monoclonal antibody which competes with the abovemonoclonal antibody in the binding of the extracellular region of CD4, amonoclonal antibody which binds to the same epitope as an epitope in anextracellular region of CD4 to which the above monoclonal antibodybinds.

Moreover, an antibody or the antibody fragment which binds to CD4 andhas apoptosis-inducing activity.

The hybridoma can be prepared, for example, by preparing the above cellexpressing CD4 as an antigen, inducing an antibody-producing cell havingantigen specificity from an animal immunized with the antigen, andfusing the antigen-producing cell with a myeloma cell. The anti-CD4monoclonal antibody can be obtained by culturing the hybridoma oradministering the hybridoma cell into an animal to cause ascites tumorin the animal and separating and purifying the culture or the ascites.

The animal immunized with an antigen may be any animal, so long as ahybridoma can be prepared, and mouse, rat, hamster, chicken, rabbit orthe like is suitably used. Also, the antibody of the present inventionincludes an antibody produced by a hybridoma obtained by fusion of thecell having antibody-producing activity can be obtained from such ananimal, and immune in vitro with a myeloma cell.

In the present invention, the recombinant antibody includes an antibodyproduced by gene recombination, such as a human chimeric antibody, ahumanized antibody (complementarity determining region (hereinafterreferred to as CDR)-grafted antibody), a human antibody and an antibodyfragment thereof. Among the recombinant antibodies, one havingcharacters of a monoclonal antibody, low immunogenecity and prolongedhalf-life in blood is preferable as a therapeutic agent. Examples of therecombinant antibody include an antibody in which the above monoclonalantibody of the present invention is modified by gene recombinationtechnology.

CH of the recombinant antibody of the resent invention is preferably ofhuman origin and includes CH1 domain, CH2 domain and CH3 domain.

Fc region of the recombinant antibody of the present invention mayinclude one or more amino acid modification, so long as it has bindingactivity to FcγR.

The human chimeric antibody is an antibody comprising a heavy chainvariable region VH and a light chain variable region VL of an antibodyof a non-human animal and CH and CL of a human antibody. Specifically,the human chimeric antibody of the present invention can be produced byobtaining cDNAs encoding VH and VL from a hybridoma which produces amonoclonal antibody which specifically recognizes CD4 and binds to theextracellular region, inserting each of them into an expression vectorfor animal cell comprising DNAs encoding CH and CL of human antibody tothereby construct a vector for expression of human chimeric antibody,and then introducing the vector into an animal cell to express theantibody.

As the CH of the human chimeric antibody, any CH can be used, so long asit belongs to human immunoglobulin (hereinafter referred to as “hIg”),and those belonging to the hIgG class are preferred, and any one of thesubclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3 andhIgG4, can be used. As the CL of the human chimeric antibody, any CL canbe used, so long as it belongs to the hIg class, and those belonging tox class or X class can be used.

Specific examples of the human chimeric antibody of the presentinvention include a human chimeric antibody wherein VH of the antibodycomprises the amino acid sequence represented by SEQ ID NO:16 and VL ofthe antibody comprises the amino acid sequence represented by SEQ IDNO:26; a human chimeric antibody wherein VH of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:12 and VL of theantibody comprises the amino acid sequence represented by SEQ ID NO:22;a human chimeric antibody wherein VH of the antibody comprises the aminoacid sequence represented by SEQ ID NO:13 and VL of the antibodycomprises the amino acid sequence represented by SEQ ID NO:23; a humanchimeric antibody wherein VH of the antibody comprises the amino acidsequence represented by SEQ ID NO:14 and VL of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:24; a human chimericantibody wherein VH of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:15 and VL of the antibody comprises the aminoacid sequence represented by SEQ ID NO:25; and the like.

In addition, examples of the chimeric antibody of the present inventioninclude a chimeric antibody which competes with the above chimericantibody in the binding of the extracellular region of CD4 and achimeric antibody which binds to the same epitope as an epitope in anextracellular region of CD4 to which the above chimeric antibody binds.

A humanized antibody is an antibody in which amino acid sequences ofCDRs of VH and VL of an antibody derived from a non-human animal aregrafted into appropriate positions of VH and VL of a human antibody. Thehumanized antibody of the present invention can be produced byconstructing cDNAs encoding a V region in which the amino acid sequencesof CDRs of VH and VL of an antibody derived from a non-human animalproduced by a hybridoma which produces a monoclonal antibody whichspecifically recognizes CD4 and binds to the extracellular region aregrafted into frame work region (hereinafter referred to as “FR”) of VHand VL of any human antibody, inserting each of them into a vector forexpression of animal cell comprising genes encoding CH and CL of a humanantibody to thereby construct a vector for expression of humanizedantibody, and introducing it into an animal cell to thereby express andproduce the humanized antibody.

As the CH of the humanized antibody, any CH can be used, so long as itbelongs to the hIg class, and those of the hIgG class are preferred andany one of the subclasses belonging to the hIgG class, such as hIgG1,hIgG2, hIgG3 and hIgG4 can be used. As the CL of the humanized antibody,any CL can be used, so long as it belongs to the hIg class, and thosebelonging to the x class or X class can be used.

Examples of the humanized antibody of the present invention include ahumanized antibody wherein CDRs 1 to 3 of VH of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:51 to 53,respectively, and CDRs 1 to 3 of VL of the antibody comprise the aminoacid sequences represented by SEQ ID NOs:54 to 56, respectively; ahumanized antibody wherein CDRs 1 to 3 of VH of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:27 to 29,respectively, and CDRs 1 to 3 of VL of the antibody comprise the aminoacid sequences represented by SEQ ID NOs:30 to 32, respectively; ahumanized antibody wherein CDRs 1 to 3 of VH of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:33 to 35,respectively, and CDRs 1 to 3 of VL of the antibody comprise the aminoacid sequences represented by SEQ ID NOs:36 to 38, respectively; ahumanized antibody wherein CDRs 1 to 3 of VH of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:39 to 41,respectively, and CDRs 1 to 3 of VL of the antibody comprise the aminoacid sequences represented by SEQ ID NOs:42 to 44, respectively; ahumanized antibody wherein CDRs 1 to 3 of VH of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:45 to 47,respectively, and CDRs 1 to 3 of VL of the antibody comprise the aminoacid sequences represented by SEQ ID NOs:48 to 50, respectively; and thelike.

Moreover, specific examples of the humanized antibody of the presentinvention include the following humanized antibodies:

with regard to the amino acid sequence of VH of the antibody, ahumanized antibody wherein VH of the antibody has an amino acid sequencein which Leu at position 18, Val at position 93, Ala at position 97 andThr at position 114 in the amino acid sequence represented by SEQ IDNO:77 are substituted with other amino acid residues,

preferably, a humanized antibody wherein VH of the antibody has an aminoacid sequence in which Val at position 93, Ala at position 97 and Thr atposition 114 in the amino acid sequence represented by SEQ ID NO:77 aresubstituted with other amino acid residues,

preferably, a humanized antibody wherein VH of the antibody has an aminoacid sequence in which Leu at position 18, Ala at position 97 and Thr atposition 114 in the amino acid sequence represented by SEQ ID NO:77 aresubstituted with other amino acid residues,

preferably, a humanized antibody having an amino acid sequence in whichAla at position 97 and Thr at position 114 are substituted with otheramino acid residues, and the like.

The amino acid sequence of VH of the antibody obtained by the aboveamino acid modifications include an amino acid sequence in which atleast one modification selected from among amino acid modifications forsubstituting Leu at position 18 with Met, Val at position 93 with Thr,Ala at position 97 with Thr, and Thr at position 114 with Ile isintroduced in the amino acid sequence represented by SEQ ID NO:77.

A specific example of the amino acid sequence of VH in which fourmodifications are introduced include an amino acid sequence in whichsubstitutions of Leu at position 18 with Met, Val at position 93 withThr, Ala at position 97 with Thr, and Thr at position 114 with Ile areintroduced in the amino acid sequence represented by SEQ ID NO:77.

Specific examples of the amino acid sequence of VH in which threemodifications are introduced in the amino acid sequence represented bySEQ ID NO:77 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 93 withThr, Ala at position 97 with Thr, and Thr at position 114 with Be areintroduced,

an amino acid sequence in which substitutions of Leu at position 18 withMet, Ala at position 97 with Thr, and Thr at position 114 with Be areintroduced,

an amino acid sequence in which substitutions of Leu at position 18 withMet, Val at position 93 with Thr, and Thr at position 114 with Be areintroduced,

an amino acid sequence in which substitutions of Leu at position 18 withMet, Val at position 93 with Thr, and Ala at position 97 with Thr areintroduced, and the like.

Specific examples of the amino acid sequence of VH in which twomodifications are introduced in the amino acid sequence represented bySEQ ID NO:77 include the following amino acid sequences:

an amino acid sequence in which substitutions of Leu at position 18 withMet, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Leu at position 18 withMet, and Ala at position 97 with Thr are introduced,

an amino acid sequence in which substitutions of Leu at position 18 withMet, and Thr at position 114 with Ile are introduced,

an amino acid sequence in which substitutions of Val at position 93 withThr, and Ala at position 97 with Thr are introduced,

an amino acid sequence in which substitutions of Val at position 93 withThr, and Thr at position 114 with Ile are introduced,

an amino acid sequence in which substitutions of Ala at position 97 withThr, and Thr at position 114 with Ile are introduced, and the like.

Specific examples of the amino acid sequence of VH in which onemodification is introduced in the amino acid sequence represented by SEQID NO:77 include the following amino acid sequences:

an amino acid sequence in which a substitution of Leu at position 18with Met is introduced,

an amino acid sequence in which a substitution of Val at position 93with Thr is introduced,

an amino acid sequence in which a substitution of Ala at position 97with Thr is introduced, and

an amino acid sequence in which a substitution of Thr at position 114with Ile is introduced.

With regard to VL of the antibody, examples include an amino acidsequence in which Ala at position 13, Val at position 15, Val atposition 19, Ala at position 47, Val at position 62, and Leu at position82 in the amino acid sequence represented by SEQ ID NO:78 aresubstituted with other amino acid residues.

Preferred is an amino acid sequence in which Ala at position 13, Val atposition 19, Val at position 62, and Leu at position 82 in the aminoacid sequence represented by SEQ ID NO:78 are substituted with otheramino acid residues.

The amino acid sequence of VL obtained by the above amino acidmodifications include an amino acid sequence in which at least onemodification selected from among amino acid modifications forsubstituting Ala at position 13 with Val, Val at position 15 with Leu,Val at position 19 with Ala, Ala at position 47 with Gln, Val atposition 62 with Ile, and Leu at position 82 with Val is introduced inthe amino acid sequence represented by SEQ ID NO:78.

Specific examples of the amino acid sequence of VL in which sixmodifications are introduced include an amino acid sequence in whichsubstitutions of Ala at position 13 with Val, Val at position 15 withLeu, Val at position 19 with Ala, Ala at position 47 with Gln, Val atposition 62 with Ile, and Leu at position 82 with Val are introduced inthe amino acid sequence represented by SEQ ID NO:78, and the like.

Specific examples of the amino acid sequence of VL in which fivemodifications are introduced in the amino acid sequence represented bySEQ ID NO:78 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 19 with Ala, Ala atposition 47 with Gln, and Val at position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 19 with Ala, Ala atposition 47 with Gln, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 19 with Ala, Val atposition 62 with Ile, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Ala at position 47 with Gln, Val atposition 62 with Ile, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, Ala at position 47 with Gln, Val atposition 62 with Be, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, Ala at position 47 with Gln, Val atposition 62 with Be, and Leu at position 82 with Val are introduced, andthe like.

Specific examples of the amino acid sequence of VL in which fourmodifications are introduced in the amino acid sequence represented bySEQ ID NO:78 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 19 with Ala, and Alaat position 47 with Gln are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 19 with Ala, and Valat position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 19 with Ala, and Leuat position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Ala at position 47 with Gln, and Valat position 62 with Be are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Ala at position 47 with Gln, and Leuat position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, Val at position 62 with Be, and Leu atposition 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, Ala at position 47 with Gln, and Valat position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, Ala at position 47 with Gln, and Leuat position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, Val at position 62 with Be, and Leu atposition 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, Ala at position 47 with Gln, and Valat position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, Ala at position 47 with Gln, and Leuat position 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, Val at position 62 with Be, and Leu atposition 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 19 withAla, Ala at position 47 with Gln, Val at position 62 with Be, and Leu atposition 82 with Val are introduced, and the like.

Specific examples of the amino acid sequence of VL in which threemodifications are introduced in the amino acid sequence represented bySEQ ID NO:78 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, and Val at position 19 with Ala areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, and Ala at position 47 with Gln areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, and Val at position 62 with Ile areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 15 with Leu, and Leu at position 82 with Val areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, and Ala at position 47 with Gln areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, and Val at position 62 with Be areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Val at position 19 with Ala, and Leu at position 82 with Val areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Ala at position 47 with Gln, and Val at position 62 with Ile areintroduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, Ala at position 47 with Gln, and Leu at position 82 with Val areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, and Ala at position 47 with Gln areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, and Val at position 62 with Be areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Val at position 19 with Ala, and Leu at position 82 with Val areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Ala at position 47 with Gln, and Val at position 62 with Ile areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Ala at position 47 with Gln, and Leu at position 82 with Val areintroduced,

an amino acid sequence in which substitutions of Val at position 19 withAla, Ala at position 47 with Gln, and Val at position 62 with Ile areintroduced,

an amino acid sequence in which substitutions of Val at position 19 withAla, Ala at position 47 with Gln, and Leu at position 82 with Val areintroduced,

an amino acid sequence in which substitutions of Ala at position 47 withGln, Val at position 62 with Be, and Leu at position 82 with Val areintroduced, and the like.

Specific examples of the amino acid sequence of VL in which twomodifications are introduced in the amino acid sequence represented bySEQ ID NO:78 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ala at position 13 withVal, and Val at position 15 with Leu are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, and Val at position 19 with Ala are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, and Ala at position 47 with Gln are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, and Val at position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Ala at position 13 withVal, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Val at position 19 with Ala are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Ala at position 47 with Gln are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Val at position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 19 withAla, and Ala at position 47 with Gln are introduced,

an amino acid sequence in which substitutions of Val at position 19 withAla, and Val at position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Val at position 19 withAla, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 47 withGln, and Val at position 62 with Ile are introduced,

an amino acid sequence in which substitutions of Ala at position 47 withGln, and Leu at position 82 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 62 withBe, and Leu at position 82 with Val are introduced, and the like.

Specific examples of the amino acid sequence of VL in which onemodification is introduced in the amino acid sequence represented by SEQID NO:78 include the following amino acid sequences:

an amino acid sequence in which a substitution of Ala at position 13with Val is introduced,

an amino acid sequence in which a substitution of Val at position 15with Leu is introduced,

an amino acid sequence in which a substitution of Val at position 19with Ala is introduced,

an amino acid sequence in which a substitution of Ala at position 47with Gln is introduced,

an amino acid sequence in which a substitution of Val at position 62with Ile is introduced,

an amino acid sequence in which a substitution of Leu at position 82with Val is introduced, and the like.

Specific example of the humanized antibody of the present inventionincludes a humanized antibody in which VH comprises the amino acidsequence represented by one selected from SEQ ID NOs:77, 96, 98 and 100,and/or VL comprises the amino acid sequence represented by one selectedfrom SEQ ID NOs:78 and 102. Furthermore, specific example of thehumanized antibody of the present invention specifically include ahumanized antibody in which H chain of variable region comprises theamino acid sequence represented by SEQ ID No:77 and/or L chain ofvariable region comprises the amino acid sequence represented by SEQ IDNo:78; a humanized antibody in which VH comprises the amino acidsequence represented by SEQ ID No:96 and/or VL comprises the amino acidsequence represented by SEQ ID No:78; a humanized antibody in which VHcomprises the amino acid sequence represented by SEQ ID No:98 and/or VLcomprises the amino acid sequence represented by SEQ ID No:78; ahumanized antibody in which VH comprises the amino acid sequencerepresented by SEQ ID No:100 and/or VL comprises the amino acid sequencerepresented by SEQ ID No:78; a humanized antibody in which VH comprisesthe amino acid sequence represented by SEQ ID No:100 and/or VL comprisesthe amino acid sequence represented by SEQ ID No:102; and the like.

In addition, examples of the humanized antibody of the present inventioninclude a humanized antibody which competes with the above humanizedantibody in the binding of the extracellular region of CD4 and ahumanized antibody which binds to the same epitope as an epitope in anextracellular region of CD4 to which the above humanized antibody binds.

A human antibody is originally an antibody naturally existing in thehuman body, and it also includes an antibody obtained from a humanantibody phage library or a human antibody-producing transgenic animal,which is prepared based on the recent advanced techniques in geneticengineering, cell engineering and developmental engineering.

The antibody existing in the human body can be prepared, for example byisolating a human peripheral blood lymphocyte, immortalizing it byinfecting with EB virus or the like and then cloning it to therebyobtain lymphocytes capable of producing the antibody, culturing thelymphocytes thus obtained, and purifying the antibody from thesupernatant of the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab and scFv are expressed on the phage surface byinserting a gene encoding an antibody prepared from a human B cell intoa phage gene. A phage expressing an antibody fragment having the desiredantigen binding activity can be recovered from the library, using itsactivity to bind to an antigen-immobilized substrate as the index. Theantibody fragment can be converted further into a human antibodymolecule comprising two full H chains and two full L chains by geneticengineering techniques.

A human antibody-producing transgenic animal is an animal in which ahuman antibody gene is integrated into cells. Specifically, a humanantibody-producing transgenic animal can be prepared by introducing agene encoding a human antibody into a mouse ES cell, grafting the EScell into an early stage embryo of other mouse and then developing it. Ahuman antibody is prepared from the human antibody-producing transgenicnon-human animal by obtaining a human antibody-producing hybridoma by ahybridoma preparation method usually carried out in non-human mammals,culturing the obtained hybridoma and forming and accumulating the humanantibody in the supernatant of the culture.

In the amino acid sequence constituting the above antibody or antibodyfragment, a monoclonal antibody or antibody fragment thereof in whichone or more amino acids are deleted, substituted, inserted or added,having activity similar to the above antibody or antibody fragment isalso included in the monoclonal antibody or antibody fragment of thepresent invention.

The number of amino acids which are deleted, substituted, insertedand/or added is one or more, and is not specifically limited, but it iswithin the range where deletion, substitution or addition is possible byknown methods such as the site-directed mutagenesis [Molecular Cloning2nd Edition, Cold Spring Harbor Laboratory Press (1989), Currentprotocols in Molecular Biology, John Wiley & Sons (1987-1997), NucleicAcids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409(1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985),Proc. Natl. Acad. Sci. USA, 82, 488 (1985)] or the like. For example,the number is 1 to dozens, preferably 1 to 20, more preferably 1 to 10,and most preferably 1 to 5.

The expression “one or more amino acids are deleted, substituted,inserted or added” in the amino acid sequence of the above antibodymeans the followings. That is, it means there is deletion, substitution,insertion or addition of one or plural amino acids at optional positionsin the same sequence and one or plural amino acid sequences. Also, thedeletion, substitution, insertion or addition may occur at the same timeand the amino acid which is substituted, inserted or added may be eithera natural type or a non-natural type. The natural type amino acidincludes L-alanine, L-asparagine, L-aspartic acid, L-glutamine,L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine,L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and thelike.

Preferable examples of mutually substitutable amino acids are shownbelow. The amino acids in the same group are mutually substitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine, cyclohexylalanineGroup B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid, 2-aminosuberic acidGroup C: asparagine, glutamineGroup D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acidGroup E: proline, 3-hydroxyproline, 4-hydroxyprolineGroup F: serine, threonine, homoserineGroup G: phenylalanine, tyrosine

The antibody fragment of the present invention includes Fab, F(ab′)₂,Fab′, scFv, diabody, dsFv, a peptide comprising CDR and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and having antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating an IgG antibody molecule with a protease, papain(cleaved at an amino acid residue at position 224 of the H chain), arebound together through a disulfide bond.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and antigen binding activity and comprising two Fab regionswhich are bound in the hinge position obtained by digesting the lowerpart of two disulfide bonds in the hinge region of IgG, with an enzyme,pepsin. The F(ab′)₂ of the present invention can be produced by treatinga monoclonal antibody which specifically recognizes CD4 and binds to theextracellular region with a protease, pepsin. Also, the F(ab′)₂ can bealso produced by binding Fab′ described below via a thioether bond or adisulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cleaving adisulfide bond at the hinge region of the above F(ab′)₂. The Fab′ of thepresent invention can be produced by F(ab′)₂ which specificallyrecognizes CD4 and binds to the extracellular region, with a reducingagent, such as dithiothreitol. Also, the Fab′ can be produced byinserting DNA encoding Fab′ fragment of the antibody into an expressionvector for prokaryote or an expression vector for eukaryote, andintroducing the vector into a prokaryote or eukaryote to express theFab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked using an appropriate peptide linker (hereinafterreferred to as “P”) and is an antibody fragment having antigen bindingactivity. The scFv of the present invention can be produced by obtainingcDNAs encoding VH and VL of a monoclonal antibody which specificallyrecognizes CD4 and binds to the extracellular region, constructing DNAencoding scFv, inserting the DNA into an expression vector forprokaryote or an expression vector for eukaryote, and then introducingthe expression vector into a prokaryote or eukaryote to express thescFv.

A diabody is an antibody fragment wherein scFv is dimerized, is anantibody fragment having divalent antigen binding activity. In thedivalent antigen binding activity, two antigens may be the same ordifferent. The diabody of the present invention can be produced byobtaining cDNAs encoding VH and VL of a monoclonal antibody whichspecifically recognizes CD4 and binds to the extracellular region,constructing DNA encoding scFv so that the length of the amino acidsequence of the peptide linker is 8 or less residues, inserting the DNAinto an expression vector for prokaryote or an expression vector foreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residueto be substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith a known methods [Protein Engineering, 7, 697 (1994)]. The dsFv ofthe present invention can be produced by obtaining cDNAs encoding VH andVL of a monoclonal antibody which specifically recognizes CD4 and bindsto the extracellular region, constructing DNA encoding dsFv, insertingthe DNA into an expression vector for prokaryote or an expression vectorfor eukaryote, and then introducing the expression vector into aprokaryote or eukaryote to express the dsFv.

A peptide comprising CDR is constituted by including one or more regionsof CDRs of VH or VL. Peptide comprising plural CDRs can be bounddirectly or via an appropriate peptide linker. The peptide comprisingCDR of the present invention can be produced by constructing DNAencoding CDRs of VH and VL of a monoclonal antibody which specificallyrecognizes CD4 and binds to the extracellular region, inserting the DNAinto an expression vector for prokaryote or an expression vector foreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the peptide. The peptide comprising CDR can alsobe produced by a chemical synthesis method such as Fmoc method or tBocmethod.

The monoclonal antibody of the present invention includes an antibodyconjugate in which a monoclonal antibody or an antibody fragment thereofwhich specifically recognizes a three-dimensional structure of anextracellular region of CD4 and binds to the extracellular region ischemically or genetically bound to a radioisotope, an agent having a lowmolecular weight, an agent having a high molecular weight, a protein, atherapeutic antibody or the like.

The antibody conjugate of the present invention can be produced bychemically conjugating a radioisotope, an agent having a low molecularweight, an agent having a high molecular weight, a protein, atherapeutic antibody or the like to the N-terminal side or C-terminalside of an H chain or an L chain of the monoclonal antibody or theantibody fragment thereof, an appropriate substituent or side chain ofthe antibody or the antibody fragment, a sugar chain in the antibody orthe antibody fragment or the like, which specifically recognizes athree-dimensional structure of an extracellular region of CD4 and bindsto the extracellular region in the present invention [AntibodyEngineering Handbook, published by Chijin Shokan (1994)].

Also, the antibody conjugate can be genetically produced by linking aDNA encoding the monoclonal antibody or the antibody fragment thereofwhich specifically recognizes three-dimensional structure of anextracellular region of CD4 and binds to the extracellular region in thepresent invention to other DNA encoding a protein or a therapeuticantibody to be conjugated, inserting the DNA into a vector forexpression, and introducing the expression vector into an appropriatehost cell.

The radioisotope includes ¹³¹I, ¹²⁵I, ⁹⁰Y, ⁶⁴Cu, ⁹⁹Tc, ⁷⁷Lu, ²¹¹At andthe like. The radioisotope can directly be conjugated with the antibodyby Chloramine-T method. Also, a substance chelating the radioisotope canbe conjugated with the antibody. The chelating agent includes1-isothiocyanate benzyl-3-methyldiethylene-triaminepentaacetic acid(MX-DTPA) and the like.

The agent having a low molecular weight includes an anti-tumor agentsuch as an alkylating agent, a nitrosourea agent, a metabolismantagonist, an antibiotic substance, an alkaloid derived from a plant, atopoisomerase inhibitor, an agent for hormonotherapy, a hormoneantagonist, an aromatase inhibitor, a P glycoprotein inhibitor, aplatinum complex derivative, an M-phase inhibitor and a kinase inhibitor[Rinsho Syuyo-gaku (Clinical Oncology), Gan to Kagaguryoho-Sha (1996)],a steroid agent such as hydrocortisone and prednisone, a nonsteroidalagent such as aspirin and indomethacin, immune-regulating agent such asaurothiomalate, penicillamine, immuno-suppressing agent such ascyclophosphamide and azathioprine, anti-inflammatory agent such asanti-histamine agent, for example, chlorpheniramine maleate andclemastine [Ensho to Kouensho-Ryoho (Inflammation and Anti-inflammationTherapy), Ishiyaku Shuppann (1982)] and the like. Examples of theantitumor agent include amifostine (Ethyol), cisplatin, dacarbazine(DTIC), dactinomycin, mecloretamin (nitrogen mustard), streptozocin,cyclophosphamide, iphosphamide, carmustine (BCNU), lomustine (CCNU),doxorubicin (adriamycin), epirubicin, gemcitabine (Gemsal),daunorubicin, procarbazine, mitomycin, cytarabine, etoposide,methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine,bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol),docetaxel (Taxotea), aldesleukin, asparaginase, busulfan, carboplatin,oxaliplatin, nedaplatin, cladribine, camptothecin,10-hydroxy-7-ethylcamptothecin (SN38), floxuridine, fludarabine,hydroxyurea, iphosphamide, idarubicin, mesna, irinotecan (CPT-11),nogitecan, mitoxantrone, topotecan, leuprolide, megestrol, melfalan,mercaptopurine, hydroxycarbamide, plicamycin, mitotane, pegasparagase,pentostatin, pipobroman, streptozocin, tamoxifen, goserelin,leuprorelin, flutamide, teniposide, testolactone, thioguanine, thiotepa,uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone,methylprednisolone, vindesine, nimustine, semustine, capecitabine,Tomudex, azacytidine, UFT, oxaliplatin, gefitinib (Iressa), imatinib(STI 571), elrotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitor,vascular endothelial growth factor receptor (VEGFR) inhibitor,fibroblast growth factor receptor (FGFR) inhibitor, epidermal growthfactor receptor (EGFR) inhibitor such as Iressa and Tarceva, radicicol,17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine,all-trans-retinoic acid, thalidomide, anastrozole, fadrozole, letrozole,exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine,mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene(Targretin), tamoxifen, dexamethasone, progestin substances, estrogensubstances, anastrozole (Arimidex), Leuplin, aspirin, indomethacin,celecoxib, azathioprine, penicillamine, gold thiomalate,chlorpheniramine maleate, chlorpheniramine, clemastine, tretinoin,bexarotene, arsenic, voltezomib, allopurinol, calicheamicin, ibritumomabtiuxetan, Targretin, ozogamine, clarithromycin, leucovorin, ifosfamide,ketoconazole, aminoglutethimide, suramin, methotrexate, maytansinoid andderivatives thereof.

The method for conjugating the agent having low molecular weight withthe antibody includes a method in which the agent and an amino group ofthe antibody are conjugated through glutaraldehyde, a method in which anamino group of the agent and a carboxyl group of the antibody areconjugated through water-soluble carbodiimide, and the like.

The agent having a high molecular weight includes polyethylene glycol(hereinafter referred to as “PEG”), albumin, dextran, polyoxyethylene,styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer,hydroxypropylmethacrylamide, and the like. By binding these compoundshaving a high molecular weight to an antibody or antibody fragment, thefollowing effects are expected: (1) improvement of stability againstvarious chemical, physical or biological factors, (2) remarkableprolongation of half life in blood, (3) disappearance of immunogenicity,suppression of antibody production, and the like [Bioconjugate Drug,Hirokawa Shoten (1993)]. For example, the method for binding PEG to anantibody includes a method in which an antibody is allowed to react witha PEG-modifying reagent [Bioconjugate Drug, Hirokawa Shoten (1993)]. ThePEG-modifying reagent includes a modifying agent of ε-amino group oflysine (Japanese Published Unexamined Patent Application No. 178926/86),a modifying agent of a carboxyl group of aspartic acid and glutamic acid(Japanese Published Unexamined Patent Application No. 23587/81), amodifying agent of a guanidino group of arginine (Japanese PublishedUnexamined Patent Application No. 117920/90) and the like.

The immunostimulator may be any natural products known asimmunoadjuvants. Examples of an agent enhancing immunogen includeβ(1→3)glucan (lentinan, schizophyllan), α-galactosylceramide and thelike.

The protein includes a cytokine or a growth factor which activates aimmunocompetent cell, such as NK cell, macrophage or neutrophil, a toxicprotein, and the like.

Examples of the cytokine or the growth factor include interferon(hereinafter referred to as “INF”)-α, INF-β, INF-γ, interleukin(hereinafter referred to as “IL”)-2, IL-12, IL-15, IL-18, IL-21, IL-23,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), macrophage-colonystimulating factor (M-CSF) and the like. The toxic protein includesricin, diphtheria toxin, ONTAK and the like, and also includes a toxicprotein wherein mutation is introduced into a protein in order tocontrol the toxicity.

The therapeutic antibody includes an antibody against an antigen inwhich apoptosis is induced by binding of the antibody, an antibodyagainst an antigen participating in formation of pathologic state oftumor, an antibody which regulates immunological function and anantibody relating to angiogenesis in the pathologic part.

The antigen in which apoptosis is induced by binding of the antibodyincludes cluster of differentiation (hereinafter “CD”) 19, CD20, CD21,CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86(B7.2), human leukocyte antigen (HLA)-Class II, epidermal growth factorreceptor (EGFR) and the like.

The antigen for the antibody which regulates immunological functionincludes CD40, CD40 ligand, B7 family molecule (CD80, CD86, CD274,B7-DC, B7-H2, B7-H3, B7-H4), ligand of B7 family molecule (CD28, CTLA-4,ICOS, PD-1, BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor(TNF) receptor family molecule (DR4, DR5, TNFR1, TNFR2), TNF-relatedapoptosis-inducing ligand receptor (TRAIL) family molecule, receptorfamily of TRAIL family molecule (TRAIL-R1, TRAIL-R2, TRAIL-R3,TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK),RANK ligand, CD25, folic acid receptor 4, cytokine [IL-1α, IL-1β, IL-4,IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β, TNFα,etc.], receptors of these cytokines, chemokine (SLC, ELC, I-309, TARC,MDC, CTACK, etc.) and receptors of these chemokines.

The antigen for the antibody which inhibits angiogenesis in thepathologic part includes vascular endothelial growth factor (VEGF),angiopoietin, fibroblast growth factor (FGF), EGF, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF), erythropoietin(EPO), TGFβ, IL-8, Ephilin, SDF-1, receptors thereof and the like.

A fusion antibody with a protein or therapeutic antibody can be producedby linking a cDNA encoding a monoclonal antibody or an antibody fragmentto a cDNA encoding the protein, constructing a DNA encoding the fusionantibody, inserting the DNA into an expression vector for prokaryote oreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the fusion antibody.

In the case where the above antibody conjugate is used for the detectingmethod, method for quantitative determination, detection reagent,reagent for quantitative determination or diagnostic agent in thepresent invention, examples of the agent to which a monoclonal antibodyor an antibody fragment thereof of the present invention whichspecifically recognizes an extracellular region of CD4 and binds to theextracellular region is bound includes a label used in routineimmunological detecting or measuring method. The label includes enzymessuch as alkaline phosphatase, peroxidase and luciferase, luminescentmaterials such as acridinium ester and lophine, fluorescent materialssuch as fluorescein isothiocyanate (FITC) and tetramethyl rhodamineisothiocyanate (RITC), and the like.

In addition, the present invention relates to a therapeutic agent for adisease relating to a CD4-positive cell which comprises a monoclonalantibody which specifically recognizes an extracellular region of CD4and also binds to the extracellular region, or an antibody fragmentthereof as an active ingredient.

The CD4-positive cell-associated disease may be any disease so long asit is a disease relating to a cell that expresses CD4, and examplesinclude cancer, autoimmune diseases, allergic diseases, and infections.

Examples of the CD4-positive cell include a CD4-positive T cell such asTh1 cell, Th2 cell, or Th17 cell, a regulatory T cell (referred to alsoas “regulatory T cell”), a γδT cell, and the like. Further, examples ofa CD4-positive cell which relates to a disease include cancer, aninflammatory disease, i.e., autoimmune disease, or an allergic disease,based on the fact that CD4 is expressed in an abnormal cell responsiblefor such a disease.

The cancer includes blood cancer, breast cancer, uterine cancer,colorectal cancer, esophageal cancer, stomach cancer, ovarian cancer,lung cancer, renal cancer, rectal cancer, thyroid cancer, uterine cervixcancer, small intestinal cancer, prostate cancer and pancreatic cancer.Preferable examples of the cancer include blood cancer, esophagealcancer, stomach cancer, colorectal cancer, liver cancer and prostatecancer. Examples of blood cancer include Cutaneous T cell lymphoma(CTCL), peripheral T cell lymphoma (PTCL), Anaplastic large celllymphoma (ALCL), other lymphoid leukemia, myeloid leukemia, multiplemyeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma.

Examples of the autoimmune disease includes rheumatoid arthritis,psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis,type I diabetes, hepatitis, myocarditis, Sjogren's syndrome, rejectionafter transplantation and the like.

Examples of the allergic disease include acute or chronic reactiveairway disease, bronchial asthma, atopic dermatitis, allergic rhinitisand the like.

Examples of the infectious disease include human immunodeficiency virus(HIV) infection and the like.

The therapeutic agent in the present invention includes a therapeuticagent comprising the above monoclonal antibody or an antibody fragmentof the present invention as an active ingredient.

The therapeutic agent comprising the antibody or antibody fragmentthereof, or conjugate thereof of the present invention may comprise onlythe antibody or antibody fragment thereof, or conjugate thereof as anactive ingredient. It is generally preferred that the therapeutic agentis prepared as a pharmaceutical preparation produced by an appropriatemethod well known in the technical field of pharmaceutics, and by mixingit with one or more pharmaceutically acceptable carriers.

It is preferred to administer the therapeutic agent by the route that ismost effective for the treatment. Examples include oral administrationand parenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration andintravenous administration is preferred.

The therapeutic agent may be in the form of spray, capsules, tablets,granules, powder, syrup, emulsion, suppository, injection, ointment,tape, and the like.

Although the dose or the frequency of administration varies depending onthe objective therapeutic effect, administration method, treatingperiod, age, body weight and the like, it is usually 10 μg/kg to 10mg/kg per day and per adult.

Further, the present invention relates to a method for immunologicallydetecting or measuring CD4, an agent for immunologically detecting ormeasuring CD4, a method for immunologically detecting or measuring acell expressing CD4, an agent for immunologically detecting or measuringa CD4-expressing cell, and an agent for diagnosing a disease relating toa CD4-positive cell, comprising a monoclonal antibody or an antibodyfragment thereof which specifically recognizes an extracellular regionof CD4 and binds to the extracellular region, as an active ingredient.

As a method for detection or determination of the amount of CD4 in thepresent invention, any known method may be included. For example, animmunological detecting or measuring method may be exemplified.

An immunological detecting or measuring method is a method in which anantibody amount or an antigen amount is detected or determined using alabeled antigen or antibody. Examples of the immunological detecting ormeasuring method are radioactive substance-labeled immunoantibody method(RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA),luminescent immunoassay, Western blotting method, physico-chemical meansand the like.

The above disease relating to CD4 can be diagnosed by detecting ormeasuring a cell expressing CD4 by using the monoclinal antibody orantibody fragment of the present invention.

For the detection of the cell expressing CD4, known immunologicaldetecting methods can be used, and an immunoprecipitation method, afluorescent cell staining method, an immune tissue staining method andthe like are preferably used. In addition, a fluorescent antibodystaining method using FMAT 8100 HTS system (Applied Biosystem) and thelike can be used.

In the present invention, the living body sample to be used fordetecting or measuring CD4 is not particularly limited, so long as ithas a possibility of containing the polypeptide, such as tissue cells,blood, blood plasma, serum, pancreatic fluid, urine, fecal matter,tissue fluid or culture fluid.

The diagnostic agent containing the monoclonal antibody or an antibodyfragment thereof, or conjugate thereof may further contain a reagent forcarrying out an antigen-antibody reaction or a reagent for detection ofthe reaction depending on the desired diagnostic method. The reagent forcarrying out the antigen-antibody reaction includes a buffer, a salt,and the like. The reagent for detection includes a reagent generallyused for the immunological detecting or measuring method, such aslabeled secondary antibody which recognizes the monoclonal antibody,antibody fragment thereof or conjugates thereof and substratecorresponding to the labeling.

A process for producing the antibody of the present invention, a methodfor treating the disease and a method for diagnosing the disease arespecifically described below.

1. Preparation Method of Monoclonal Antibody (1) Preparation of Antigen

CD4 or a cell expressing CD4 as an antigen can be obtained byintroducing an expression vector comprising cDNA encoding a full lengthof CD4 or a partial length thereof is introduced into Escherichia coli,yeast, an insect cell, an animal cell or the like. In addition, CD4 canbe purified from various human tumor cell lines, human tissue and thelike which express a large amount of CD4. The tumor cell line and thetissue can be allowed to use as antigens. Furthermore, a syntheticpeptide having a partial sequence of the CD4 can be prepared by achemical synthesis method such as Fmoc method or tBoc method and used asan antigen.

CD4 used in the present invention can be produced, for example, byexpressing a DNA encoding CD4 in a host cell using a method described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989), Current Protocols in Molecular Biology,John Wiley & Sons (1987-1997) or the like according to the followingmethod.

Firstly, a recombinant vector is prepared by inserting a full lengthcDNA comprising the region encoding CD4 into downstream of a promoter ofan appropriate expression vector. At this time, if necessary, a DNAfragment having an appropriate length containing a region encoding thepolypeptide based on the full length cDNA, and the DNA fragment may beused instead of the above full length cDNA. Next, a transformantproducing CD4 can be obtained by introducing the recombinant vector intoa host cell suitable for the expression vector.

The expression vector includes vectors which can replicate autonomouslyin the host cell to be used or vectors which can be integrated into achromosome comprising an appropriate promoter at such a position thatthe DNA encoding the polypeptide can be transcribed.

The host cell may be any one, so long as it can express the objectivegene. Examples include a microorganism which belongs to the generaEscherichia, such as Escherichia coli, yeast, an insect cell, an animalcell and the like.

When a prokaryote such as Escherichia coli is used as the host cell, itis preferred that the recombinant vector used in the present inventionis autonomously replicable in the prokaryote and comprises a promoter, aribosome binding sequence, the DNA comprising the portion encoding CD4and a transcription termination sequence. The recombinant vector is notnecessary to have a transcription termination sequence, but atranscription termination sequence is preferably set just below thestructural gene. The recombinant vector may further comprise a generegulating the promoter.

Also, the above recombinant vector is preferably a plasmid in which thespace between Shine-Dalgarno sequence (also referred to as SD sequence),which is the ribosome binding sequence, and the initiation codon isadjusted to an appropriate distance (for example, 6 to 18 nucleotides).

Furthermore, the nucleotide sequence of the DNA encoding CD4 can besubstituted with another base so as to be a suitable codon forexpressing in a host cell, thereby improve the productivity of theobjective CD4.

Any expression vector can be used, so long as it can function in thehost cell to be used. Examples of the expression vector includes pBTrp2,pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2(manufactured by Pharmacia), pSE280 (manufactured by Invitrogen),pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN),pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83),pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1[Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA,82, 4306 (1985)], pBluescript II SK(−) (manufactured by Stratagene),pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)],pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)],pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), JapanesePublished Unexamined Patent Application No. 221091/85], pGKA2 [preparedfrom Escherichia coli IGKA2 (FERM BP-6798), Japanese PublishedUnexamined Patent Application No. 221091/85], pTerm2 (U.S. Pat. No.4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), pSupex,pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX(manufactured by Pharmacia), pET system (manufactured by Novagen),pME18SFL3 and the like.

Any promoter can be used, so long as it can function in the host cell tobe used. Examples include promoters derived from Escherichia coli, phageand the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PRpromoter and T7 promoter. Also, artificially designed and modifiedpromoters, such as a promoter in which two Ptrp are linked in tandem,tac promoter, lacT7 promoter and letI promoter, can be used.

Examples of the host cell include Escherichia coli XL1-Blue, Escherichiacoli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000,Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109,Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110,Escherichia coli NY49, Escherichia coli DH5a and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into the host cell, and examplesinclude a method using a calcium ion [Proc. Natl. Acad. Sci. USA, 69,2110 (1972), methods described in Gene, 17, 107 (1982) and Molecular &General Genetics, 168, 111 (1979)].

When an animal cell is used as the host cell, an expression vector canbe used, so long as it can function in the animal cell. Examples includepcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese PublishedUnexamined Patent Application No. 22979/91; Cytotechnology, 3, 133(1990)], pAS3-3 (Japanese Published Unexamined Patent Application No.227075/90), pCDM8 [Nature, 329, 840, (1987)], pcDNAI/Amp (manufacturedby Invitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4(manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307(1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97/10354) and the like.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of immediate early (IE) gene ofcytomegalovirus (CMV), SV40 early promoter, a promoter of retrovirus, ametallothionein promoter, a heat shock promoter, SRα promoter, Molonymurine leukemia virus promoter or enhancer, and the like. Also, theenhancer of the IE gene of human CMV can be used together with thepromoter.

Examples of the host cell include human leukemia cell Namalwa cell,monkey COS cell, Chinese hamster ovary cell CHO cell (Journal ofExperimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60,1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973);Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260(1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad.Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics,Appendix I, II (pp. 883-900)), CHO/DG44, CHO-K1 (ATCC CCL-61), DUkXB11(ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3 cell, rat myeloma YB2/3HL.P2.G11.16Ag.20 (also known asYB2/0), mouse myeloma cell NSO, mouse myeloma cell SP2/0-Ag14, Syrianhamster cells BHK or HBT5637 (Japanese Published Unexamined PatentApplication No. 000299/88) and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into an animal cell, and examplesinclude electroporation [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], and the like.

CD4 can be produced by culturing the transformant derived from amicroorganism, an animal cell or the like having a recombinant vectorcomprising the DNA encoding CD4 in a medium to form and accumulate CD4in the culture, and recovering it from the culture. The method forculturing the transformant in the medium is carried out according to theusual method used in culturing of hosts.

When CD4 is expressed in a cell derived from eukaryote, CD4 to whichsugars or sugar chains bind can be obtained.

When a microorganism transformed with a recombinant vector containing aninducible promoter is cultured, an inducer can be added to the medium,if necessary. For example, isopropyl-β-D-thiogalactopyranoside or thelike can be added to the medium when a microorganism transformed with arecombinant vector using lac promoter is cultured; or indoleacrylic acidor the like can be added thereto when a microorganism transformed with arecombinant vector using trp promoter is cultured.

When a transformant obtained using an animal cell as the host cell iscultured, the medium includes generally used RPMI 1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)] and 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], Iscoove's modified Dulbecco'smedium (IMDM), the media to which fetal calf serum, etc. is added, andthe like. The culturing is carried out generally at a pH of 6 to 8 and30 to 40° C. for 1 to 7 days in the presence of 5% CO₂. If necessary, anantibiotic such as kanamycin or penicillin can be added to the mediumduring the culturing.

Regarding the expression method of the gene encoding CD4, in addition todirect expression, secretory production, fusion protein expression andthe like can be carried out according to the method described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989).

The process for producing CD4 includes a method of intracellularexpression in a host cell, a method of extracellular secretion from ahost cell, a method of producing on a host cell membrane outer envelope,and the like. The appropriate method can be selected by changing thehost cell used and the structure of the CD4 produced.

When the CD4 is produced in a host cell or on a host cell membrane outerenvelope, CD4 can be positively secreted extracellularly in accordancewith the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)],the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989),Genes Develop., 4, 1288 (1990)], the methods described in JapanesePublished Unexamined Patent Application No. 336963/93 and WO 94/23021,and the like.

Also, the production amount of CD4 can be increased in accordance withthe method described in Japanese Published Unexamined Patent ApplicationNo. 227075/90 utilizing a gene amplification system using adihydrofolate reductase gene.

The resulting CD4 can be isolated and purified, for example, as follows.

When CD4 is intracellularly expressed in a dissolved state, the cellsafter culturing are recovered by centrifugation, suspended in an aqueousbuffer and then disrupted using ultrasonicator, French press, MantonGaulin homogenizer, dynomill or the like to obtain a cell-free extract.The cell-free extract is centrifuged to obtain a supernatant, and apurified preparation can be obtained by subjecting the supernatant to ageneral enzyme isolation and purification techniques such as solventextraction; salting out with ammonium sulfate etc.; desalting;precipitation with an organic solvent; anion exchange chromatographyusing a resin such as diethylaminoethyl (DEAE)-sepharose, DIAION HPA-75(manufactured by Mitsubishi Chemical); cation exchange chromatographyusing a resin such as S-Sepharose FF (manufactured by Pharmacia);hydrophobic chromatography using a resin such as butyl-Sepharose orphenyl-Sepharose; gel filtration using a molecular sieve; affinitychromatography; chromatofocusing; electrophoresis such as isoelectricfocusing; and the like which may be used alone or in combination.

When CD4 is expressed intracellularly by forming an inclusion body, thecells are recovered, disrupted and centrifuged in the same manner, andthe inclusion body of CD4 are recovered as a precipitation fraction. Therecovered inclusion body of the protein is solubilized with a proteindenaturing agent. The protein is made into a normal three-dimensionalstructure by diluting or dialyzing the solubilized solution, and then apurified preparation of CD4 is obtained by the same isolationpurification method as above.

When CD4 or the derivative such as a glycosylated product is secretedextracellularly, CD4 or the derivative such as a glycosylated productcan be recovered from the culture supernatant. That is, the culture istreated by a method such as centrifugation in the same manner as aboveto obtain a culture supernatant, a purified preparation of CD4 can beobtained from the culture supernatant by the same isolation purificationmethod as above.

Also, CD4 used in the present invention can be produced by a chemicalsynthesis method, such as Fmoc method or tBoc method. Also, it can bechemically synthesized using a peptide synthesizer manufactured byAdvanced ChemTech, Perkin-Elmer, Pharmacia, Protein TechnologyInstrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, or thelike.

(2) Immunization of Animal and Preparation of Antibody-Producing Cellfor Fusion

A mouse, rat or hamster 3 to 20 weeks old is immunized with the antigenprepared in the above (1), and antibody-producing cells are collectedfrom the spleen, lymph node or peripheral blood of the animal. Also,when the increase of a sufficient titer in the above animal isrecognized due to low immunogenecity, a CD4 knockout mouse may by usedas an animal to be immunized.

The immunization is carried out by administering the antigen to theanimal through subcutaneous, intravenous or intraperitoneal injectiontogether with an appropriate adjuvant (for example, complete Freund'sadjuvant, combination of aluminum hydroxide gel with pertussis vaccine,or the like). When the antigen is a partial peptide, a conjugate isproduced with a carrier protein such as BSA (bovine serum albumin), KLH(keyhole limpet hemocyanin) or the like, which is used as the antigen.

The administration of the antigen is carried out 5 to 10 times every oneweek or every two weeks after the first administration. On the 3rd to7th day after each administration, a blood sample is collected from thefundus of the eye, the reactivity of the serum with the antigen istested, for example, by enzyme immunoassay [Antibodies—A LaboratoryManual, Cold Spring Harbor Laboratory (1988)] or the like. An animalshowing a sufficient antibody titer in their sera against the antigenused for the immunization is used as the supply source ofantibody-producing cells for fusion.

Three to seven days after final administration of the antigen, tissuecontaining the antibody-producing cells such as the spleen from theimmunized animal is excised to collect the antibody-producing cells.When the spleen cells are used, the spleen is cut out and loosened,followed by centrifuged. Then, antibody-producing cells for fusion areobtained by removing erythrocytes.

(3) Preparation of Myeloma Cell

An established cell line obtained from mouse is used as myeloma cells.Examples include 8-azaguanine-resistant mouse (derived from BALB/c)myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiologyand Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J.Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269 (1978)],P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], P3-X63-Ag8 (X63)[Nature, 256, 495 (1975)] and the like.

The myeloma cells are subcultured in a normal medium [a medium in whichglutamine, 2-mercaptoethanol, gentamicin, FBS and 8-azaguanine are addedto RPMI1640 medium] and they are subcultured in the normal medium 3 or 4days before cell fusion to ensure the cell number of 2×10⁷ or more onthe day for fusion.

(4) Cell Fusion and Preparation of Hybridoma for Producing MonoclonalAntibody

The antibody-producing cells for fusion obtained by the above (2) andmyeloma cells obtained by the above (3) were sufficiently washed with aminimum essential medium (MEM) or PBS (1.83 g of disodium hydrogenphosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of sodiumchloride, 1 liter of distilled water, pH 7.2) and mixed to give a ratioof the antibody-producing cells: the myeloma cells=5 to 10:1, followedby centrifugation. Then, the supernatant is discarded. The precipitatedcell group is sufficiently loosened. After loosening the precipitatedcell, the mixture of polyethylene glycol-1000 (PEG-1000), MEM anddimethylsulfoxide is added to the cell under stirring at 37° C. Inaddition, 1 to 2 mL of MEM medium is added several times every one ortwo minutes, and MEM is added to give a total amount of 50 mL. Aftercentrifugation, the supernatant is discarded. After the cells are gentlyloosen, the cells are gently suspended in HAT medium [a medium in whichhypoxanthine, thymidine and aminopterin is added to the normal medium].The suspension is cultured in a 5% CO₂ incubator for 7 to 14 days at 37°C.

After the culturing, a portion of the culture supernatant is sampled anda hybridoma which is reactive to an antigen containing CD4 and is notreactive to an antigen not containing CD4 is selected by binding assayas described below. Then, cloning is carried out twice by a limitingdilution method [Firstly, HT medium (HAT medium from which aminopterinis removed) is used, and secondly, the normal medium is used], and ahybridoma which shows a stably high antibody titer is selected as themonoclonal antibody-producing hybridoma.

(5) Preparation of Purified Monoclonal Antibody

The hybridoma cells producing a monoclonal antibody obtained by theabove (4) are administered by intraperitoneal injection into 8- to10-week-old mice or nude mice treated with 0.5 mL of pristane(2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by feeding for 2 weeks). The hybridoma developsascites tumor in 10 to 21 days. The ascitic fluid is collected from themice, centrifuged to remove solids, subjected to salting out with 40 to50% ammonium sulfate and then precipitated by caprylic acid, passedthrough a DEAE-Sepharose column, a protein A column or a gel filtrationcolumn to collect an IgG or IgM fraction as a purified monoclonalantibody.

Furthermore, a monoclonal antibody-producing hybridoma obtained by theabove (4) is cultured in RPMI1640 medium containing FBS or the like andthe supernatant is removed by centrifugation. The precipitated cells aresuspended in Hybridoma SFM medium containing 5% DIGO GF21 and culturedfor 3 to 7 days. The purified monoclonal antibody can be obtained bycentrifusing the obtained cell suspension, followed by purifying theresulting supernatant with Protein A column or Protein G column tocollect the IgG fractions.

The subclass of the antibody can be determined using a subclass typingkit by enzyme immunoassay. The amount of the protein can be determinedby the Lowry method or from the absorbance at 280 nm.

(6) Selection of Monoclonal Antibody

Selection of monoclonal antibody is carried out by the following bindingassay using an enzyme immunoassay method and kinetic analysis withBiacore.

(6-a) Binding Assay

As the antigen, a gene-introduced cell or a recombinant protein obtainedby introducing an expression vector containing a cDNA encoding CD4obtained in (1) into Escherichia coli, yeast, an insect cell, an animalcell or the like, or a purified polypeptide or partial peptide obtainedfrom a human tissue is used. When the antigen is a partial peptide, aconjugate is prepared with a carrier protein such as BSA or KLH and isused.

After making these antigens into a solid layer by dispensing in a96-well plate, a substance to be tested such as serum, a culturesupernatant of a hybridoma or a purified monoclonal antibody isdispensed therein as the primary antibody and allowed to react. Afterthoroughly washing with PBS or PBS-Tween, an anti-immunoglobulinantibody labeled with biotin, an enzyme, a chemiluminescent material, aradiation compound or the like is dispensed therein as the secondaryantibody and allowed to react. After thoroughly washing with PBS-Tween,the reaction is carried out in response to the label of the secondaryantibody to select a monoclonal antibody which specifically reacts withthe antigen.

The antibody which competes with the anti-CD4 monoclonal antibody of thepresent invention can be prepared by adding an antibody to be tested tothe above-mentioned binding assay system and carrying out reaction. Thatis, a monoclonal antibody which competes with the thus obtainedmonoclonal antibody for its binding to the extracellular region of CD4can be prepared by carrying out a screening of an antibody by which thebinding of the monoclonal antibody is inhibited when the antibody to betested is added.

Furthermore, an antibody which binds to an epitope which is the same asthe epitope recognized by the monoclonal antibody which binds to theextracellular region of CD4 of the present invention can be obtained byidentifying the epitope of the antibody obtained in the above bindingassay, and preparing a partial synthetic peptide, a synthetic peptidemimicking the conformational structure of the epitope or the like,followed by immunization.

(6-b) Kinetic Analysis with Biacore

The kinetics between an antigen and a test substance is measured usingBiacore T100 and then the obtained results are analyzed using analysissoftware accompanied with the apparatus. After anti-IgG mouse antibodyis immobilized onto to the a CM 5 sensor chip by an amine couplingmethod, a test substance such as culture supernatant of a hybridoma, apurified antibody is allowed to flow, bind at an appropriate amount, andfurther flow an antigen at plural known concentrations, followed bymeasuring the binding and dissociation. Using the obtained data and thesoftware accompanied with the apparatus, the kinetics analysis iscarried out using the 1:1 binding model to obtain necessary parameters.Otherwise, after human CD4 is immobilized onto the sensor chip by anamino coupling method, a purified monoclonal antibody is allowed to flowat plural known concentrations followed by measuring the binding anddissociation. Using the obtained data and the software accompanied withthe apparatus, the kinetics analysis is carried out using bivalentanalyte model to obtain necessary parameters.

2. Preparation of Recombinant Antibody

As production examples of recombinant antibodies, processes forproducing a human chimeric antibody and a humanized antibody are shownbelow.

(1) Construction of Vector for Expression of Recombinant Antibody

A vector for expression of recombinant antibody is an expression vectorfor animal cell into which DNAs encoding CH and CL of a human antibodyhave been inserted, and is constructed by cloning each of DNAs encodingCH and CL of a human antibody into an expression vector for animal cell.

The C region of a human antibody may be CH and CL of any human antibody.Examples include CH belonging to γ1 subclass, CL belonging to κ class,and the like. As the DNAs encoding CH and CL of a human antibody, thecDNA may be generally used and a chromosomal DNA comprising an exon andan intron can be also used. As the expression vector for animal cell,any expression vector can be used, so long as a gene encoding the Cregion of a human antibody can be inserted thereinto and expressedtherein. Examples include pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103[J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR[Proc. Natl. Acad. Sci. USA, 78, 1527 (1980], pSG1bd2-4 [Cytotechnol.,4, 173 (1990)], pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)] and thelike. Examples of a promoter and enhancer used for an expression vectorfor animal cell include an SV40 early promoter [J. Biochem., 101, 1307(1987)], a Moloney mouse leukemia virus LTR [Biochem. Biophys. Res.Commun., 149, 960 (1987)], an immunoglobulin H chain promoter [Cell, 41,479 (1985)] and enhancer [Cell, 33, 717 (1983)] and the like.

The vector for expression of recombinant antibody may be either of atype in which a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In respect of easiness ofconstruction of a vector for expression of recombinant antibody,easiness of introduction into animal cells, and balance between theexpression amounts of antibody H and L chains in animal cells, a tandemtype of the vector for expression of recombinant antibody is morepreferred [J. Immunol. Methods, 167, 271 (1994)]. Examples of the tandemtype of the vector for expression of recombinant antibody includepKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], and thelike.

(2) Obtaining of cDNA Encoding V Region of Antibody Derived fromNon-Human Animal and Analysis of Amino Acid Sequence

Obtaining of cDNAs encoding VH and VL of a non-human animal antibody andanalysis of amino acid sequence are carried out as follows.

mRNA is extracted from hybridoma cells producing an antibody derivedfrom a non-human animal to synthesize cDNA. The synthesized cDNA iscloned into a vector such as a phage or a plasmid, to prepare a cDNAlibrary. Each of a recombinant phage or recombinant plasmid containingcDNA encoding VH or VL is isolated from the library using DNA encoding apart of the C region or V region of a mouse antibody as the probe. Thefull length of the nucleotide sequences of VH and VL of a mouse antibodyderived from a non-human animal of interest on the recombinant phage orrecombinant plasmid are determined, and the full length of the aminoacid sequences of VH and VL are deduced from the nucleotide sequences,respectively.

Examples of the non-human animal for preparing a hybridoma cell whichproduces a non-human antibody include mouse, rat, hamster, rabbit or thelike. Any animals can be used so long as a hybridoma cell can beproduced therefrom.

Examples of the method for preparing total RNA from a hybridoma cellinclude a guanidine thiocyanate-cesium trifluoroacetate method [Methodsin Enzymol., 154, 3 (1987)], the use of a kit such as RNA easy kit(manufactured by Qiagen) and the like.

Examples of the method for preparing mRNA from total RNA include anoligo (dT) immobilized cellulose column method [Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989)], a method using a kit such as Oligo-dT30 <Super> mRNAPurification Kit (manufactured by Takara Bio) and the like. Also,examples of a kit for preparing mRNA from a hybridoma cell include FastTrack mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNAPurification Kit (manufactured by Pharmacia) and the like.

Examples of the method for synthesizing cDNA and preparing a cDNAlibrary include known methods [Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Lab. Press (1989); Current Protocols in MolecularBiology, Supplement 1, John Wiley & Sons (1987-1997)]; a method using akit such as Super Script Plasmid System for cDNA Synthesis and PlasmidCloning (manufactured by GIBCO BRL), ZAP-cDNA Kit (manufactured byStratagene), etc.; and the like.

The vector into which the synthesized cDNA using mRNA extracted from ahybridoma cell as the template is inserted for preparing a cDNA librarymay be any vector, so long as the cDNA can be inserted. Examples includeZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [NucleicAcids Research, 17, 9494 (1989)], λzapII (manufactured by Stratagene),λgt10 and λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)],Lambda BlueMid (manufactured by Clontech), λExCell and pT7T3 18U(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)],pUC18 [Gene, 33, 103 (1985)], and the like.

Any Escherichia coli for introducing the cDNA library constructed by aphage or plasmid vector may be used, so long as the cDNA library can beintroduced, expressed and maintained. Examples include XL1-Blue MRF′[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088 andY1090 [Science, 222: 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)],K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)], andthe like.

A colony hybridization or plaque hybridization method using an isotope-or fluorescence-labeled probe may be used for selecting cDNA clonesencoding VH and VL of a non-human antibody or the like from the cDNAlibrary [Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989)].

Also, the cDNAs encoding VH and VL can be prepared through polymerasechain reaction (hereinafter referred to as “PCR”; Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley& Sons (1987-1997)) by preparing primers and using cDNA prepared frommRNA or a cDNA library as the template.

The nucleotide sequence of the cDNA can be determined by digesting thecDNA selected with appropriate restriction enzymes and the like, cloningthe fragments into a plasmid such as pBluescript SK(−) (manufactured byStratagene), carrying out the reaction by a usually used nucleotideanalyzing method. For example, a nucleotide analyze is carried out byusing an automatic nucleotide sequence analyzer such as ABI PRISM3700(manufactured by PE Biosystems) and A.L.F. DNA sequencer (manufacturedby Pharmacia) after a reaction such as the dideoxy method [Proc. Natl.Acad. Sci. USA, 74, 5463 (1977)].

Whether the obtained cDNAs encode the full amino acid sequences of VLand VL of the antibody containing a secretory signal sequence can beconfirmed by estimating the full length of the amino acid sequences ofVH and VL from the determined nucleotide sequence and comparing themwith the full length of the amino acid sequences of VH and VL of knownantibodies [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)]. The length of the secretory signalsequence and N-terminal amino acid sequence can be deduced by comparingthe full length of the amino acid sequences of VH and VL of the antibodycomprising a secretory signal sequence with full length of the aminoacid sequences of VH and VL of known antibodies [Sequences of Proteinsof Immunological Interest, US Dept. Health and Human Services (1990],and the subgroup to which they belong can also be known. Furthermore,the amino acid sequence of each of CDRs of VH and VL can be found bycomparing the obtained amino acid sequences with amino acid sequences ofVH and VL of known antibodies [Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991)].

Moreover, the novelty of the full length of the amino acid sequence ofVH and VL can be examined by carrying out a homology search withsequences in any database, for example, SWISS-PROT, PR-Protein or thelike using the obtained full length of the amino acid sequences of VHand VL, for example, according to the BLAST method [J. Mol. Biol., 215,403 (1990)] or the like.

(3) Construction of Vector for Expression of Human Chimeric Antibody

cDNA encoding each of VH and VL of antibody of non-human animal iscloned in the upstream of genes encoding CH or CL of human antibody ofvector for expression of recombinant antibody mentioned in the above (1)to thereby construct a vector for expression of human chimeric antibody.

For example, in order to ligate cDNA comprising a nucleotide sequence of3′-terminal of VH or VL of antibody of non-human animal and a nucleotidesequence of 5′-terminal of CH or CL of human antibody, each cDNAencoding VH and VL of antibody of non-human animal is prepared so as toencodes appropriate amino acids encoded by a nucleotide sequence of alinkage portion and designed to have an appropriate recognition sequenceof a restriction enzyme. The obtained cDNAs encoding VH and VL ofantibody are respectively cloned so that each of them is expressed in anappropriate form in the upstream of gene encoding CH or CL of humanantibody of the vector for expression of humanized antibody mentioned inthe above (1) to construct a vector for expression of human chimericantibody.

In addition, cDNA encoding VH or VL of a non-human animal antibody isamplified by PCR using a synthetic DNA having a recognition sequence ofan appropriate restriction enzyme at both ends and each of them iscloned to the vector for expression of recombinant antibody obtained inthe above (1).

(4) Construction of cDNA Encoding V Region of Humanized Antibody

cDNAs encoding VH or VL of a humanized antibody can be obtained asfollows.

Amino acid sequences of framework region (hereinafter referred to as“FR”) in VH or VL of a human antibody to which amino acid sequences ofCDRs in VH or VL of an antibody derived from a non-human animal antibodyare transplanted are respectively selected. Any amino acid sequences ofFR of a human antibody can be used, so long as they are derived fromhuman. Examples include amino acid sequences of FRs of human antibodiesregistered in database such as Protein Data Bank or the like, and aminoacid sequences common to subgroups of FRs of human antibodies [Sequencesof Proteins of Immunological Interest, US Dept. Health and HumanServices (1990], and the like. In order to inhibit the decrease in thebinding activity of the antibody, amino acid sequences having highhomology (at least 60% or more) with the amino acid sequence of FR in VHor VL of the original antibody is selected.

Then, amino acid sequences of CDRs of the original antibody are graftedto the selected amino acid sequence of FR in VH or VL of the humanantibody, respectively, to design each amino acid sequence of VH or VLof a humanized antibody. The designed amino acid sequences are convertedto DNA sequences by considering the frequency of codon usage found innucleotide sequences of genes of antibodies [Sequence of Proteins ofImmunological Interest, US Dept. Health and Human Services (1990], andthe DNA sequence encoding the amino acid sequence of VH or VL of ahumanized antibody is designed.

Based on the designed nucleotide sequences, several synthetic DNAshaving a length of about 100 nucleotides are synthesized, and PCR iscarried out using them. In this case, it is preferred that 6 syntheticDNAs per each of the H chain and the L chain are designed in view of thereaction efficiency of PCR and the lengths of DNAs which can besynthesized.

Furthermore, the cDNA encoding VH or VL of a humanized antibody can beeasily cloned into the vector for expression of humanized antibodyconstructed in (1) by introducing the recognition sequence of anappropriate restriction enzyme to the 5′ terminal of the synthetic DNAsexisting on the both ends.

Otherwise, it can be carried out using a synthetic DNA as one DNAencoding each of the full-length H chain and the full-length L chainbased on the designed DNA sequence.

After the PCR, an amplified product is cloned into a plasmid such aspBluescript SK (−) (manufactured by Stratagene) or the like, and thenucleotide sequence is determined according to a method similar to themethod described in (2) to obtain a plasmid having a DNA sequenceencoding the amino acid sequence of VH or VL of a desired humanizedantibody.

(5) Modification of Amino Acid Sequence of V Region of HumanizedAntibody

It is known that when a humanized antibody is produced by simplygrafting only CDRs in VH and VL of an antibody derived from a non-humananimal into FRs of VH and VL of a human antibody, its antigen bindingactivity is lower than that of the original antibody derived from anon-human animal [BIO/TECHNOLOGY, 9, 266 (1991)]. In humanizedantibodies, among the amino acid sequences of FRs in VH and VL of ahuman antibody, an amino acid residue which directly relates to bindingto an antigen, an amino acid residue which interacts with an amino acidresidue in CDR, and an amino acid residue which maintains thethree-dimensional structure of an antibody and indirectly relates tobinding to an antigen are identified and modified to an amino acidresidue which is found in the original non-humanized antibody to therebyincrease the antigen binding activity which has been decreased.

In order to identify the amino acid residues relating to the antigenbinding activity in FR, the three-dimensional structure of an antibodyis constructed and analyzed by X-ray crystallography [J. Mol. Biol.,112, 535 (1977)], computer-modeling [Protein Engineering, 7, 1501(1994)] or the like. In addition, various attempts must be currently benecessary, for example, several modified antibodies of each antibody areproduced and the correlation between each of the modified antibodies andits antibody binding activity is examined.

The modification of the amino acid sequence of FR in VH and VL of ahuman antibody can be accomplished using various synthetic DNA formodification according to PCR as described in (4). With regard to theamplified product obtained by the PCR, the nucleotide sequence isdetermined according to the method as described in (2) so that whetherthe objective modification has been carried out is confirmed.

(6) Construction of Vector for Expression of Humanized Antibody

A vector for expression of humanized antibody can be constructed bycloning each cDNA encoding VH or VL of a constructed recombinantantibody into upstream of each gene encoding CH or CL of the humanantibody in the vector for expression of recombinant antibody asdescribed in (1).

For example, when recognizing sequences of an appropriate restrictionenzymes are introduced to the 5′-terminal of synthetic DNAs positionedat both ends among synthetic DNAs used in the construction of VH or VLof the humanized antibody in (4) and (5), cloning can be carried out sothat they are expressed in an appropriate form in the upstream of eachgene encoding CH or CL of the human antibody in the vector forexpression of a humanized antibody as described in (1).

(7) Transient Expression of Recombinant Antibody

In order to efficiently evaluate the antigen binding activity of varioushumanized antibodies produced, the recombinant antibodies can beexpressed transiently using the vector for expression of humanizedantibody as described in (3) and (6) or the modified expression vectorthereof.

Any cell can be used as a host cell, so long as the host cell canexpress a recombinant antibody. Generally, COS-7 cell (ATCC CRL1651) isused in view of its high expression amount [Methods in Nucleic AcidsRes., CRC Press, 283 (1991)].

Examples of the method for introducing the expression vector into COS-7cell include a DEAE-dextran method [Methods in Nucleic Acids Res., CRCPress, 283 (1990], a lipofection method [Proc. Natl. Acad. Sci. USA, 84,7413 (1987)], and the like.

After introduction of the expression vector, the expression amount andantigen binding activity of the recombinant antibody in the culturesupernatant can be determined by the enzyme immunoassay [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific(1987)] and the like.

(8) Obtaining Transformant which Stably Expresses Recombinant Antibodyand Preparation of Recombinant Antibody

A transformant which stably expresses a recombinant antibody can beobtained by introducing the vector for expression of recombinantantibody described in (3) and (6) into an appropriate host cell.

Examples of the method for introducing the expression vector into a hostcell include electroporation [Japanese Published Unexamined PatentApplication No. 257891/90, Cytotechnology, 3, 133 (1990)] and the like.

As the host cell into which a vector for expression of a recombinantantibody is introduced, any cell can be used, so long as it is a hostcell which can produce the recombinant antibody. Examples include CHO-K1(ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S(Life Technologies, Cat #11619), rat myeloma cell YB2/3HL.P2.G11.16Ag.20(also referred to as YB2/0), mouse myeloma cell NSO, mouse myeloma cellSP2/0-Ag14 (ATCC No. CRL1581), mouse P3×63-Ag8.653 cell (ATCC No.CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafterreferred to as “dhfr”) is defective [Proc. Natl. Acad. Sci. U.S.A., 77,4216 (1980)], lection resistance-acquired Lec13 [Somatic Cell andMolecular genetics, 12, 55 (1986)], CHO cell in whichα1,6-fucosyltransaferse gene is defected (WO 2005/35586, WO 02/31140),rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC No. CRL1662), and the like.

In addition, host cells in which activity of a protein such as an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,a protein such as an enzyme relating to the modification of a sugarchain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in a complex typeN-glycoside-linked sugar chain, or a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced is decreased or deleted, preferably CHO cell in whichα1,6-fucosyltransferase gene is defected as described in WO05/35586,WO02/31140 or the like, can also be used.

After introduction of the expression vector, transformants which expressa recombinant antibody stably are selected by culturing in a medium foranimal cell culture containing an agent such as G418 sulfate(hereinafter referred to as “G418”) or the like (Japanese PublishedUnexamined Patent Application No. 257891/90).

Examples of the medium for animal cell culture include RPMI1640 medium(manufactured by Invitrogen), GIT medium (manufactured by NihonPharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium(manufactured by Invitrogen), Hybridoma-SFM medium (manufactured byInvitrogen), media obtained by adding various additives such as fetalcalf serum (hereinafter referred to as “FCS”) to these media, and thelike. The recombinant antibody can be produced and accumulated in aculture supernatant by culturing the selected transformants in a medium.The expression amount and antigen binding activity of the recombinantantibody in the culture supernatant can be measured by ELISA or thelike. Also, in the transformant, the expression amount of therecombinant antibody can be increased by using DHFR amplification systemor the like according to the method disclosed in Japanese PublishedUnexamined Patent Application No. 257891/90.

The recombinant antibody can be purified from the culture supernatant ofthe transformant by using a protein A column [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988)]. For example, the recombinant antibody can be purified by acombination of gel filtration, ion-exchange chromatography,ultrafiltration and the like.

The molecular weight of the H chain or the L chain of the purifiedrecombinant antibody or the antibody molecule as a whole is determinedby polyacrylamide gel electrophoresis (hereinafter referred to as“SDS-PAGE”) [Nature, 227, 680 (1970)], Western blotting [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988)], and the like.

3. Activity Evaluation of the Monoclonal Antibody or Antibody Fragment

The activity of the purified monoclonal antibody or antibody fragment ofthe present invention can be evaluated in the following manner.

The binding activity to CD4-expressing cell is evaluated by the bindingassay described in the above 1-(6-a) and a surface plasmon resonancemethod using such as the Biacore system described in the above (6-b).Furthermore, it can be measured by fluorescent antibody technique[Cancer Immunol. Immunother., 36, 373 (1993)], a surface plasmonresonance method using such as BIAcore system or the like. Furthermore,it can be measured by fluorescent antibody technique [Cancer Immunol.Immunother., 36, 373 (1993)].

In addition, CDC activity or ADCC activity against an antigen positivecell line is evaluated by a known method [Cancer Immunol. Immunother.,36, 373 (1993)].

4. Method of Controlling Effector Activity of Antibody

As a method for controlling an effector activity of the anti-CD4monoclonal antibody of the present invention, a method for controllingan amount of fucose (hereinafter, referred to also as “core fucose”)which is bound in α-1,6 linkage to N-acetylglucosamine (GlcNAc) presentin a reducing end of a complex type N-linked sugar chain which is boundto asparagine (Asn) at position 297 of an Fc region of an antibody(WO2005/035586, WO2002/31140, and WO00/61739), a method for controllingan effector activity of a monoclonal antibody by modifying amino acidgroup(s) of an Fc region of the antibody, and the like are known. Theeffector activity of the anti-CD4 monoclonal antibody of the presentinvention can be controlled by using any of the methods.

The “effector activity” means an antibody-dependent activity which isinduced via an Fc region of an antibody. As the effector activity, anantibody-dependent cellular cytotoxicity (ADCC activity), acomplement-dependent cytotoxicity (CDC activity), an antibody-dependentphagocytosis (ADP activity) by phagocytic cells such as macrophages ordendritic cells, and the like are known.

By controlling a content of core fucose of a complex type N-linked sugarchain of Fc of an antibody, an effector activity of the antibody can beincreased or decreased. According to a method for lowering a content offucose which is bound to a complex type N-linked sugar chain bound to Fcof the antibody, an antibody to which fucose is not bound can beobtained by the expression of an antibody using a CHO cell which isdeficient in a gene encoding α1,6-fucosyltransferase. The antibody towhich fucose is not bound has a high ADCC activity. On the other hand,according to a method for increasing a content of fucose which is boundto a complex type N-linked sugar chain bound to Fc of an antibody, anantibody to which fucose is bound can be obtained by the expression ofan antibody using a host cell into which a gene encodingα1,6-fucosyltransferase is introduced. The antibody to which fucose isbound has a lower ADCC activity than the antibody to which fucose is notbound.

Further, by modifying amino acid residue(s) in an Fc region of anantibody, the ADCC activity or CDC activity can be increased ordecreased. The ADCC activity can be controlled by increasing ordecreasing the binding activity to FcγR due to the modification(s) ofamino acid residue(s) in an Fc region. In addition, the CDC activity canbe controlled by increasing or decreasing the binding activity ofcomplement due to the modification(s) of amino acid residue(s) in an Fcregion. For example, the binding activity to an antibody can beincreased by using the amino acid sequence of the Fc region described inUS2007/0148165. Further, the ADCC activity or CDC activity can beincreased or decreased by modifying the amino acid as described in U.S.Pat. No. 6,737,056, or 7,297,775 or WO2005/070963.

Furthermore, an antibody in which the effector activity is controlledcan be obtained by combining the above method for controlling a sugarchain and the method for modifying amino acid(s) in an Fc region.

5. Method for Treating Disease Using the Anti-CD4 Monoclonal Antibody orAntibody Fragment of the Present Invention

A monoclonal antibody which specifically recognizes a nativethree-dimensional structure of CD4 and binds to the extracellularregion, or an antibody fragment thereof of the present invention can beused for treating a disease relating to CD4.

Examples of a route of administration include oral administration andparenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration. In the caseof an antibody or peptide formulation, intravenous administration ispreferred. Examples of the dosage form includes sprays, capsules,tablets, powder, granules, syrups, emulsions, suppositories, injections,ointments, tapes and the like.

The pharmaceutical preparation suitable for oral administration includesemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced using,as additives, water; sugars such as sucrose, sorbitol and fructose;glycols such as polyethylene glycol and propylene glycol; oils such assesame oil, olive oil and soybean oil; antiseptics such asp-hydroxybenzoic acid esters; flavors such as strawberry flavor andpeppermint; and the like.

Capsules, tablets, powders, granules and the like can be produced using,as additives, excipients such as lactose, glucose, sucrose and mannitol;disintegrating agents such as starch and sodium alginate; lubricantssuch as magnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropylcellulose and gelatin; surfactants such as fatty acidester; plasticizers such as glycerin; and the like.

The pharmaceutical preparation suitable for parenteral administrationincludes injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, aglucose solution or a mixture of both thereof.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Sprays can be prepared using the antibody or antibody fragment as suchor using it together with a carrier which does not stimulate the buccalor airway mucous membrane of the patient and can facilitate absorptionof the compound by dispersing it as fine particles. The carrier includeslactose, glycerol and the like. It is possible to produce pharmaceuticalpreparations such as aerosols and dry powders.

In addition, the components exemplified as additives for oralpreparations can also be added to the parenteral preparations.

5. Method for Diagnosing Disease Using the Anti-CD4 Monoclonal Antibodyor Antibody Fragment of the Present Invention

A disease relating to CD4 can be diagnosed by detecting or determiningCD4 or a cell expressing CD4 using the monoclonal antibody or antibodyfragment of the present invention.

A diagnosis of cancer, one of the diseases relating to CD4, can becarried out by, for example, the detection or measurement of CD4 asfollows.

The diagnosis of cancer can be carried out by detecting CD4 expressingon the cell in a patient's body by an immunological method such as aflow cytometer.

An immunological method is a method in which an antibody amount or anantigen amount is detected or determined using a labeled antigen orantibody. Examples of the immunological method include radioactivesubstance-labeled immunoantibody method, enzyme immunoassay, fluorescentimmunoassay, luminescent immunoassay, Western blotting method,physico-chemical means and the like.

Examples of the radioactive substance-labeled immunoantibody methodinclude a method, in which the antibody or antibody fragment of thepresent invention is allowed to react with an antigen, a cell expressingan antigen or the like, then anti-immunoglobulin antibody subjected to aradioactive labeling or a binding fragment thereof is allowed to reacttherewith, followed by determination using a scintillation counter orthe like.

Examples of the enzyme immunoassay include a method, in which theantibody or antibody fragment of the present invention is allowed toreact with an antigen, a cell expressing an antigen or the like, then ananti-immunoglobulin antibody or an binding fragment thereof subjected toantibody labeling is allowed to react therewith and the colored pigmentis measured by a spectrophotometer, and, for example, sandwich ELISA maybe used. As a label used in the enzyme immunoassay, any known enzymelabel (Enzyme Immunoassay, published by Igaku Shoin, 1987) can be usedas described already. Examples include alkaline phosphatase labeling,peroxidase labeling, luciferase labeling, biotin labeling and the like.

Sandwich ELISA is a method in which an antibody is bound to a solidphase, antigen to be detected or measured is trapped and anotherantibody is allowed to react with the trapped antigen. In the ELISA, twokinds of antibody which recognizes the antigen to be detected ormeasured or the antibody fragment thereof in which antigen recognizingsite is different are prepared and the first antibody or antibodyfragments is previously adsorbed on a plate (such as a 96-well plate)and the second antibody or antibody fragment is labeled with afluorescent substance such as FITC, an enzyme such as peroxidase, orbiotin. The plate to which the above antibody is adsorbed is allowed toreact with the cell separated from living body or disrupted cellsuspension thereof, tissue or disintegrated solution thereof, culturedcells, serum, pleural effusion, ascites, eye solution or the like, thenallowed to react with a labeled monoclonal antibody or an antibodyfragment and a detection reaction corresponding to the labeled substanceis carried out. The antigen concentration in the sample to be tested canbe calculated from a calibration curve prepared by a stepwise dilutionof antigen of known concentration. As antibody used for sandwich ELISA,any of polyclonal antibody and monoclonal antibody may be used orantibody fragments such as Fab, Fab′ and F(ab)₂ may be used. As acombination of 2 kinds of antibodies used in sandwich ELISA, acombination of monoclonal antibodies or antibody fragments recognizingdifferent epitopes may be used or a combination of polyclonal antibodywith monoclonal antibody or antibody fragments may be used.

A fluorescent immunoassay includes a method described in the literatures[Monoclonal Antibodies—Principles and practice, Third Edition, AcademicPress (1996); Manual for Monoclonal Antibody Experiments, KodanshaScientific (1987)] and the like. As a label for the fluorescentimmunoassay, any of known fluorescent labels [Fluorescent Immunoassay,by Akira Kawao, Soft Science, (1983)] may be used as described already.Examples of the label include FITC, RITC and the like.

The luminescent immunoassay can be carried out using the methodsdescribed in the literature [Bioluminescence and Chemical Luminescence,Rinsho Kensa, 42, Hirokawa Shoten (1998)] and the like. As a label usedfor luminescent immunoassay, any of known luminescent labels can beincluded. Examples include acridinium ester, lophine or the like may beused.

Western blotting is a method in which an antigen or a cell expressing anantigen is fractionated by SDS-polyacrylamide gel electrophoresis[Antibodies—A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)],the gel is blotted onto PVDF membrane or nitrocellulose membrane, themembrane is allowed to react with antigen-recognizing antibody orantibody fragment, further allowed to react with an anti-mouse IgGantibody or antibody fragment which is labeled with a fluorescentsubstance such as FITC, an enzyme label such as peroxidase, a biotinlabeling, or the like, and the label is visualized to confirm thereaction. An example thereof is described below. Cells or tissues inwhich a polypeptide having the amino acid sequence represented by SEQ IDNO:2 is expressed are dissolved in a solution and, under reducingconditions, 0.1 to 30 μg as a protein amount per lane is electrophoresedby an SDS-PAGE method. The electrophoresed protein is transferred to aPVDF membrane and allowed to react with PBS containing 1 to 10% of BSA(hereinafter referred to as “BSA-PBS”) at room temperature for 30minutes for blocking. Here, the monoclonal antibody of the presentinvention is allowed to react therewith, washed with PBS containing 0.05to 0.1% Tween 20 (hereinafter referred to as “Tween-PBS”) and allowed toreact with goat anti-mouse IgG labeled with peroxidase at roomtemperature for 2 hours. It is washed with Tween-PBS and a band to whichthe monoclonal antibody is bound is detected using ECL Western BlottingDetection Reagents (manufactured by Amersham) or the like to therebydetect a polypeptide having the amino acid sequence represented by SEQID NO:2. As an antibody used for the detection in Western blotting, anantibody which can be bound to a polypeptide having no three-dimensionalstructure of a natural type is used.

The physicochemical method is specifically carried out by reacting CD4as the antigen with the antibody or antibody fragment of the presentinvention to form an aggregate, and detecting this aggregate. Otherexamples of the physicochemical methods include a capillary method, aone-dimensional immunodiffusion method, an immunoturbidimetry, a lateximmunoturbidimetry [Handbook of Clinical Test Methods, Kanehara Shuppan,(1988)] and the like. For example, in a latex immunodiffusion method, acarrier such as polystyrene latex having a particle size of about of 0.1to 1 μm sensitized with antibody or antigen may be used and when anantigen-antibody reaction is carried out using the corresponding antigenor antibody, scattered light in the reaction solution increases whiletransmitted light decreases. When such a change is detected asabsorbance or integral sphere turbidity, it is now possible to measureantigen concentration, etc. in the sample to be tested.

For the detection of the cell expressing CD4, known immunologicaldetection methods can be used, and an immunoprecipitation method, aimmuno cell staining method, an immune tissue staining method, afluorescent antibody staining method and the like are preferably used.

An immunoprecipitation method is a method in which a cell expressing CD4is allowed to react with the monoclonal antibody or antibody fragment ofthe present invention and then a carrier having specific binding abilityto immunoglobulin such as protein G-Sepharose is added so that anantigen-antibody complex is precipitated. Also, the following method canbe carried out.

The above-described antibody or antibody fragment of the presentinvention is solid-phased on a 96-well plate for ELISA and then blockedwith BSA-PBS. When the antibody is in a non-purified state such as aculture supernatant of hybridoma cell, anti-mouse immunoglobulin or ratimmunoglobulin or protein A or Protein G or the like is previouslyadsorbed on a 96-well plate for ELISA and blocked with BSA-PBS and aculture supernatant of hybridoma cell is dispensed thereto for binding.After BSA-PBS is discarded and the residue is sufficiently washed withPBS, reaction is carried out with a dissolved solution of cells ortissues expressing CD4. An immune precipitate is extracted from thewell-washed plate with a sample buffer for SDS-PAGE and detected by theabove-described Western blotting.

An immune cell staining method or an immune tissue staining method are amethod where cells or tissues in which antigen is expressed are treated,if necessary, with a surfactant, methanol or the like to make anantibody easily permeate to the cells or tissues, then the monoclonalantibody of the present invention is allowed to react therewith, thenfurther allowed to react with an anti-immunoglobulin antibody or bindingfragment thereof subjected to fluorescent labeling such as FITC, enzymelabel such as peroxidase or biotin labeling and the label is visualizedand observed under a microscope. In addition, cells of tissues can bedetected by an immunofluorescent staining method where cells are allowedto react with a fluorescence-labeled antibody and analyzed by a flowcytometer [Monoclonal Antibodies—Principles and practice, Third Edition,Academic Press (1996), Manual for Experiments of Monoclonal Antibodies,Kodansha Scientific (1987)] in which cells are allowed to react with afluorescence-labeled antibody and analyzed by a flow cytometer.Particularly, the monoclonal antibody or antibody fragment of thepresent invention which binds to an extracellular region of the CD4 candetect a cell expressing the polypeptide maintaining a natural typethree-dimensional structure.

In addition, in the case of using FMAT8100HTS system (manufactured byApplied Biosystems) and the like among fluorescent antibody stainingmethods, the antigen quantity or antibody quantity can be measuredwithout separating the formed antibody-antigen complex and the freeantibody or antigen which is not concerned in the formation of theantibody-antigen complex.

The present invention is described below by Examples; however, thepresent invention is not limited to the following Examples.

Example 1 Construction of CD4 Transfectant

(1) Construction of CD4 Expression Vector

A vector into which a human CD4 nucleotide sequence was inserted wasconstructed as follows, from a vector (Clone No. 5226427, hereinafterreferred to as “pCMV-CD4”, manufactured by Open Biosystems) where ahuman CD4 gene was inserted into a vector pCMV-SPORT6.

The CD4 gene fragment was amplified using pCMV-CD4 as a template andprimers comprising the nucleotide sequences represented by SEQ ID NOs:79and 80, in accordance with a conventional PCR method. The PCR productwas purified by a PCR Purification Kit (manufactured by Qiagen), andthen treated with restriction enzymes EcoRI and SpeI. Similarly, avector pBluescript II sk(−) (manufactured by Stratagene) (hereinafter,referred to as “pBS”) was treated with restriction enzymes EcoRI andSpeI. The resulting two fragments were ligated using a Ligation high(manufactured by Toyobo) in accordance with the instructions attachedthereto. An Escherichia coli DH5α strain (manufactured by Toyobo) wastransformed with the obtained recombinant plasmid DNA solution. Aplasmid DNA was prepared from the clone of the transformant and wasconfirmed by treatment with restriction enzymes, and a plasmid pBS/CD4into which a CD4 gene was inserted was obtained. The resulting pBS/CD4into which a CD4 gene was inserted, and an expression vector pKANTEX93(WO97/10354) for an animal cell were treated with restriction enzymesEcoRI and SpeI. The reaction liquid was fractionated by agarose gelelectrophoresis, thereby recovering an EcoRI-SpeI fragment of CD4 and anEcoRI-SpeI fragment of pKANTEX93, respectively. These two fragments wereligated using a Ligation high (manufactured by Toyobo) in accordancewith the instructions attached thereto. An Escherichia coli DH5α strain(manufactured by Toyobo) was transformed with the resulting recombinantplasmid DNA solution. A plasmid DNA was prepared from the clone of thetransformant and was confirmed by the treatment with restrictionenzymes, and a plasmid pKANTEX/CD4 into which a CD4 gene was insertedwas obtained. The plasmid obtained was allowed to react using a BigDyeTerminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PEBiosystems) in accordance with the instructions attached thereto, andthen its nucleotide sequence was analyzed by a sequencer of the samecompany, ABI PRISM3700. As a result, a vector pKANTEX/CD4 into which acDNA encoding human CD4 was cloned was obtained. FIG. 1 illustrates aschematic diagram for the construction of the vector.

(2) Construction of CD4-Expressing CHO/DG44 cell

In accordance with electroporation [Cytotechnology, 3, 133 (1990)], theCD4 expression vector pKANTEX/CD4 constructed in Section (1) into aCHO/DG44 cell [Somatic Cell and Molecular Genetics, 12, 555 (1986)] wasintroduced in the following manner. The cells used herein are thosesubcultured in a medium where 1×HT supplement (manufactured byInvitrogen) was added to IMDM (manufactured by Invitrogen) supplementedwith 10% fetal bovine serum (manufactured by Life Technologies) andgentamicin (50 μg/mL, manufactured by Nacalai Tesque) (hereinafter,referred to as “A3 medium”). CHO/DG44 cells were suspended in a buffercontaining potassium chloride (137 nmol/L), sodium chloride (2.7nmol/L), disodium hydrogen phosphate (8.1 mmol/L), sodium dihydrogenphosphate (1.5 nmol/L) and magnesium chloride (4 mmol/L) (hereinafter,referred to as “K-PBS”) to give a cell concentration of 8×10⁶ cells/mL,and the resulting cell suspension (200 μL, 1.6×10⁶ cells in terms ofcell count) was mixed with the expression plasmid pKANTEX/CD4 (8 μg).The mixture was transferred into a cuvette (interelectrode distance: 2mm), followed by gene transfer using a GenePulser II (manufactured byBio-Rad) at a pulse voltage of 0.35 kV and an electric capacity of 250μF. After the cuvette was allowed to stand on ice, the cell suspensionin the cuvette was suspended in a cell culture vessel containing A3medium and cultured in a 5% CO₂ incubator at 37° C. Thereafter, thecells were cultured in a medium containing G418 (manufactured byInvitrogen, 0.5 mg/mL), and then a transformed cell line resistant toG418 was obtained. In addition, a clone showing a high expression levelof CD4 was selected by adding methotrexate to the medium and increasingthe concentration in a stepwise manner. In this manner, a CD4-expressingCHO cell line (hereinafter, referred to as “CHO/CD4 cell” or “CD4transfectant”) was obtained.

Example 2 Construction of CD4-Fc

(1) Construction of CD4-Fc Expression Vector

The vector pBS/CD4 constructed in Example 1(1), into which a human CD4gene was inserted, and a vector pKANTEX93 were mixed for use as atemplate. PCR was carried out using the mixture as a template, and foursynthetic oligo nucleotides represented by SEQ ID NOs:79, 80, 81, and 82as primers. According to this reaction, a nucleotide sequence in which agene encoding an extracellular region of a CD4 protein was ligated witha gene of the Fc region was synthesized (hereinafter, referred to as“CD4-Fc gene”). The resulting PCR product was fractionated by agarosegel electrophoresis, and a desired CD4-Fc gene (about 2 kbp) was cleavedand purified. PCR was carried out again using the purified product as atemplate, and two primers represented by SEQ ID NOs:79 and 82, so thatthe CD4-Fc gene was amplified. The resulting PCR product wasfractionated by agarose gel electrophoresis, and a desired gene CD4-Fc(about 2 kbp) was cleaved and treated with restriction enzymes EcoRI andSpeI, thereby obtaining an EcoRI-SpeI fragment of the CD4-Fc gene.Similarly, the vector pKANTEX93 was treated with restriction enzymesEcoRI and SpeI. The resulting two fragments were ligated using aLigation High (manufactured by Toyobo). An Escherichia coli DH5α strain(manufactured by Toyobo) was transformed with the obtained recombinantplasmid DNA solution. A plasmid DNA was prepared from the clone of thetransformant and was confirmed by treatment with restriction enzymes,and a plasmid pKANTEX/CD4-Fc into which the CD4-Fc gene was inserted wasobtained. The plasmid obtained was reacted using a BigDye TerminatorCycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems)in accordance with the instructions attached thereto, and then thenucleotide sequence was analyzed by a sequencer of the same company, ABIPRISM3700. As a result, a vector pKANTEX/CD4-Fc into which a cDNAencoding CD4-Fc was cloned was obtained. FIG. 2 illustrates a schematicdiagram for the construction of the vector.

(2) Expression of CD4-Fc

Using the pKANTEX/CD4-Fc constructed in Section (1), a CHO cellexpressing CD4-Fc (hereinafter, referred to as “CHO/CD4-Fc”) wasobtained by a conventional method [Antibody Engineering, A PracticalGuide, W.H. Freeman and Company (1992)].

(3) Purification of Soluble CD4-Fc

After culturing the CHO/CD4-Fc constructed in Section (2) under the sameculture conditions as in Example 1(2), the cell suspension was recoveredand centrifuged at 3000 rpm and 4° C. for 20 minutes to recover theculture supernatant, and then the culture supernatant wasfiltration-sterilized using a 0.22-μm pore size Millex GV filter(manufactured by Millipore). The CD4-Fc protein was purified from theresulting culture supernatant, using a Protein A High-capacity resin(manufactured by Millipore) column in accordance with the instructionsattached thereto.

Example 3 Preparation of Anti-CD4 Monoclonal Antibody

(1) Preparation of Immunogen

In order to obtain an anti-CD4 monoclonal antibody, a lyophilizedproduct of recombinant human CD4 (catalogue number: 514-CD/CF,manufactured by R&D Systems) dissolved in a Dulbecco's phosphate buffer(phosphate buffered saline: PBS), or a human CD4-Fc fusion protein(hereinafter, referred to as “CD4-Fc”) prepared in Example 2(3) was usedas an immunogen.

(2) Immunization of Animal and Preparation of Antibody-Producing Cell

Into SD rats (manufactured by Japan SLC) was administered 20 μg of therecombinant human CD4 or the CD4-Fc prepared in Example 2(3), togetherwith 2 mg of an aluminum hydroxide adjuvant (Antibodies—A LaboratoryManual, Cold Spring Harbor Laboratory, p 99, 1988) and 1×10⁹ cells of apertussis vaccine (manufactured by Chiba Serum Institute). Two weeksafter the administration, 20 μg of the CD4 and 2 mg of an aluminumhydroxide adjuvant alone were administered to the rats once a week, fourtimes in total. Blood was partially collected from the fundus oculi veinof the rats, and the serum antibody titer was confirmed by CD4 bindingELISA. The spleen was extracted from a rat which showed a sufficientantibody titer three days after the final immunization. The spleen wasminced into small pieces in Minimum Essential Medium (MEM, manufacturedby Nissui Pharmaceutical Co., Ltd.), ground and centrifuged (1200 rpm, 5minutes). Tris-ammonium chloride buffer (pH 7.6) was added to treat theresulting precipitation fraction for 1 minute at 37° C., whereby redblood cells were removed. The resulting precipitation fraction (cellfraction) was washed three times with MEM, and used in the cell fusion.

(3) CD4 Binding ELISA

For the assay a 96-well ELISA plate (manufactured by Greiner) was used.For this purpose, 2 μg/mL of the recombinant human CD4 prepared inExample 1(1) was dispensed at a concentration of 50 μL/well to theplate, and allowed to stand at 4° C. overnight for adsorption. After theplate was washed with PBS, 100 μL/well of 1% bovine serum albumin(BSA)-PBS (hereinafter, referred to as “BSA-PBS”) was added to theplate, and the plate was then allowed to stand at room temperature for 1hour such that the remaining active groups were blocked. Then, BSA-PBSwas discarded, and 50 μL/well of an appropriate dilution of theimmunized rat serum as a primary antibody was dispensed to the plate,and the plate was then allowed to stand for 2 hours. The plate waswashed with 0.05% polyoxyethylene (20) sorbitan monolaurate [(theequivalent of ICI trade name Tween 20, manufactured by Wako PureChemical)]/PBS (hereinafter referred to as “Tween-PBS”), and 50 μL/wellof a peroxidase-labeled goat anti-rat IgG (H+L chain) antibody(manufactured by Zymed) as a secondary antibody was added to the plate,and the plate was then allowed to stand at room temperature for 1 hour.The plate was washed with Tween-PBS, and color-developed by adding 50μL/well of a 2,2-azinobis(3-ethylbenzothiazole-6-sulfonic acid)ammonium(ABTS) substrate solution [1 mmoL/L ABTS, 0.1 moL/L citrate buffer (pH4.2), 0.1% H₂O₂]. The reaction was stopped by adding 50 μL/well of a 5%sodium lauryl sulfate (SDS) solution, and an absorbance (OD415 nm) wasthen measured using a plate reader Emax (Molecular Devices).

(4) Preparation of Mouse Myeloma Cell

The 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P3-U1,purchased fromATCC) was cultured in RPMI 1640 medium (manufactured byInvitrogen) supplemented with 10% of fetal bovine serum to ensure thecell count of 2×10⁷ or more necessary for cell fusion, and was providedas parent cells in the cell fusion.

(5) Measurement of Culture Supernatant by FMAT Method

The CD4 transfectants prepared in Example 1 or CHO/DG44 cells as anegative control were seeded onto a 96-well FMAT plate (manufactured byApplied Biosystems) at a cell density of 1×10⁴ cells/50 μL/well,followed by overnight culture in a 5% CO2 incubator at 37° C. To theplate, 10 μL/well of the culture supernatant of the hybridoma was addedand then 50 μL/well of an ALEXA 647-labeled goat anti-rat IgG (H+L)antibody (manufactured by Invitrogen) diluted with BSA-PBS wasimmediately added thereto, followed by allowing to stand at roomtemperature under shading for 3 hours. Thereafter, a wavelength of 650to 685 nm which was excited with a 633 nm He/Ne laser was measured by an8200 Cellular Detection System (manufactured by Applied Biosystems).Hereinafter, this measurement method is referred to as FMAT method.

(6) Construction of Hybridoma

Mouse spleen cells obtained in Section Example 3(2) and myeloma cellsobtained in Example 3(4) were mixed at a ratio of 10:1 and centrifugedat 1,200 rpm for 5 minutes. The cell group of the resultingprecipitation fraction was well loosened. A mixture of 1 g ofpolyethylene glycol-1000 (PEG-1000), 1 mL of Minimum Essential Medium(hereinafter, referred to as “MEM”) and 0.35 mL of dimethyl sulfoxide(DMSO) was added thereto at 0.5 mL/1×10⁸ mouse spleen cells and at 37°C. under stirring, and 1 mL of MEM was added several times to thesuspension every 1 minute. Then, MEM was added to give a total volume of50 mL. The cell suspension was centrifuged (900 rpm, 5 minutes), andcells of the resulting precipitation fraction were slowly loosened andthen slowly suspended in 100 mL of HAT medium [HAT Media Supplement(manufactured by Invitrogen) was added to RPMI 1640 medium supplementedwith 10% fetal bovine serum]. The suspension was dispensed into a96-well culture plate at 200 μL/well and cultured in a 5% CO₂ incubatorat 37° C. for 10 to 14 days. After the culture, the culture supernatantwas measured according to the FMAT method described in Example 3(5), anda well which was reactive to the CHO/CD4 prepared in Example 1 and wasnot reactive to the CHO/DG44 cell was selected. Then, the selected cellsproducing a CD4-specific antibody were subjected to cloning by alimiting dilution method twice to establish hybridomas KM4065, KM4066,KM4067, KM4068 and KM4069 which produce anti-CD4 rat monoclonalantibodies.

Example 4 Activity Evaluation for Anti-CD4 Rat Monoclonal Antibody

(1) Reactivity of Anti-CD4 Rat Monoclonal Antibody for HumanCD4-Positive Cell Line by FCM

Cells were blocked by adding 1 mg/mL of human IgG (manufactured bySigma) to 1 to 5×10⁵ cells of the human T cell lymphoma cell lineHPB-ALL. Anti-CD4 rat monoclonal antibodies KM4065 to KM4069 and ananti-CD4 mouse antibody OKT4 (manufactured by BioLegend) wereappropriately diluted with BSA-PBS and added thereto to give a totalvolume of 50 μL. These cell suspensions were allowed to react on ice for60 minutes, and the cells were washed two times with BSA-PBS. To thecells, 50 μL of Alexa-488-labeled anti-rat IgG (H+L) goat antibody(manufactured by Invitrogen) diluted with BSA-PBS was added, followed byreaction on ice for 40 minutes. The cells were washed once with BSA-PBSand suspended in BSA-PBS, and the fluorescence intensity was measured bya flow cytometer (manufactured by Beckman Coulter).

FIG. 3 shows the mean fluorescence intensity (MFI value) when themonoclonal antibody was reacted at a concentration of 5, 0.5, and 0.05μg/mL. It was demonstrated that all of the anti-CD4 rat monoclonalantibodies KM4065 to KM4069, and the anti-CD4 mouse antibody OKT4 bindto the CD4-positive cell line in an antibody concentration-dependentmanner.

(2) Binding Activity of Anti-CD4 Rat Monoclonal Antibody to RecombinantHuman CD4 by Biacore

In order to kinetically analyze the binding activity of anti-CD4 ratmonoclonal antibodies KM4065 to KM4069 and commercially availableanti-human CD4 mouse antibody for recombinant human CD4, the bindingactivity was measured by surface plasmon resonance method (SPR). All ofthe following manipulations were carried out using the Biacore T100(manufactured by GE Healthcare Bio-Sciences). The anti-mouse IgGantibody was immobilized on a CM5 sensor chip (manufactured by GEHealthcare Bio-Sciences) by an amine coupling method using a MouseAntibody Capture Kit (manufactured by GE Healthcare Bio-Sciences) inaccordance with the protocols attached thereto. Anti-CD4 rat monoclonalantibodies KM4065 to KM4069, Leu-3a (BD Biosciences), OKT4 (BioLegend)and 13B8.2 (Beckman Coulter) were added to be captured on the anti-mouseIgG antibody-immobilized chip to give 100 RU (resonance unit),respectively. Thereafter, the recombinant human CD4 (manufactured byR&D) diluted from 2500 ng/mL in five steps was allowed to run at a flowrate of 30 μL/min onto the chip, and the sensorgram corresponding toeach concentration was obtained. The analysis was carried out in the 1:1binding model using an analysis software attached to the apparatus,thereby calculating an association rate constant ka and a dissociationrate constant kd of individual antibodies for the human CD4.

As a result, an association rate constant (hereinafter, referred to as“ka”), a dissociation rate constant (hereinafter, referred to as “kd”)and a dissociation constant K_(D) (kd/ka) of individual antibodies thusobtained are given in Table 2.

As shown in Table 2, the anti-CD4 rat monoclonal antibodies KM4065 andKM4066 exhibited a higher affinity than the conventional antibody.

TABLE 2 Antibody ka (1/Ms) kd (1/s) K_(D) (M) KM4065 3.57E+05 1.52E−064.26E−12 KM4066 1.34E+06 1.18E−04 8.82E−11 KM4067 8.75E+05 4.73E−045.41E−10 KM4068 1.85E+06 2.04E−03 1.10E−09 KM4069 1.21E+06 5.84E−044.82E−10 Leu-3a* 1.10E+06 1.29E−04 1.17E−10 OKT4** 1.05E+06 3.06E−032.90E−09 13B8.2*** 4.44E+05 1.56E−04 3.52E−10 *manufactured by BDBioscience, **manufactured by Biolegend, ***manufactured by BeckmanCoulter

(3) Evaluation of Complement-Dependent Cellular Cytotoxicity (CDCActivity) of Anti-CD4 Rat Monoclonal Antibody on CD4 Transfectant

The CDC activity of the anti-CD4 rat monoclonal antibodies KM4066 toKM4068 and the anti-CD4 mouse antibody OKT4 on the CD4 transfectantconstructed in Example 1 was measured using a culture supernatant of therat hybridoma, according to the following procedure.

The CD4 transfectant was washed with PBS, washed with RPMI 1640 medium(manufactured by Wako Pure Chemical) containing 10% fetal bovine serum(FBS), and prepared to give an optimum concentration in the same medium,which was then used as a target cell suspension. The target cellsuspension was dispensed into a 96-well flat-bottom plate (manufacturedby Sumitomo Bakelite) at 5×10⁴ cells/well. In addition, an anti-CD4chimeric antibody solution prepared to give an appropriate concentrationand a human complement (manufactured by Sigma) at a final concentrationof 25% were added thereto to give a total volume of 100 μL/well. Inaddition, a reaction well not containing the antibody (0% cellularcytotoxicity well) as a negative control, and a reaction well notcontaining the cell (100% cellular cytotoxicity well) as a positivecontrol were respectively prepared. The reaction was carried out in a 5%CO₂ incubator at 37° C. for 3 hours. After the reaction was complete, 10μL/well of a WST-1 reagent (manufactured by Roche Diagnostics) was addedto the reaction wells, followed by reaction at 37° C. for 3 hours. Anabsorbance at 450 nm (OD450) for each well was measured using a platereader Emax (manufactured by Molecular Devices). From the absorbance ofeach well, the CDC activity (cellular cytotoxicity [%]) was calculatedaccording to the following formula.

CDC activity (cellular cytotoxicity [%])=100×{1−(absorbance of reactionwell-absorbance of 100% lysis well)/(absorbance of 0% lysiswell−absorbance of 100% lysis well)}  (Formula)

The measurement results are shown in FIG. 4. As shown in FIG. 4, theconventional anti-CD4 mouse antibody OKT4 had a CDC activity, andsimilarly all the clones of the anti-CD4 rat monoclonal antibodiesKM4066 to KM4068 obtained in the present invention also exhibited a CDCactivity.

Example 5 Isolation and Analysis of cDNA Encoding Variable Region ofAnti-CD4 Monoclonal Antibody

(1) Preparation of mRNA from Anti-CD4 Monoclonal Antibody-ProducingHybridoma Cell

From 5×10⁷ to 1×10⁸ cells of the respective hybridomas KM4065 to KM4069obtained in Example 3, mRNA of the respective anti-CD4 rat monoclonalantibodies was prepared using RNAeasy Mini kit (manufactured by Qiagen)and Oligotex™-dT30 <Super> mRNA Purification Kit (manufactured byTakara) in accordance with the instructions attached thereto.

(2) Gene Cloning of H Chain and L Chain Variable Regions of Anti-CD4Monoclonal Antibody

Using a SMART RACE cDNA Amplification Kit (manufactured by Clontech) inaccordance with the instructions attached thereto, a cDNA was obtainedfrom 0.5 μg of mRNA of the monoclonal antibody obtained in Example 5(1).cDNA fragments of heavy chain variable regions (hereinafter referred toas “VH”) of the respective antibodies were amplified by carrying out PCRusing the thus obtained cDNA as a template, and using a universal primerAmix attached to the kit and one selected from a rat IgG1-specificprimer (SEQ ID NO:3), a rat IgG2a-specific primer (SEQ ID NO:4), and anIgG2b-specific primer (SEQ ID NO:5). Also, cDNA fragments of light chainvariable regions (hereinafter, referred to as “VL”) of the respectiveantibodies were amplified by carrying out PCR using a rat Ig(κ)-specificprimer (SEQ ID NO:6) instead of the respective subclass-specific primersof the antibody. PCR for any antibody subclass was carried out byheating at 94° C. for 5 minutes; 5 cycles each consisting of reaction at94° C. for 15 seconds and reaction at 72° C. for 3 minutes; 5 cycleseach consisting of reaction at 94° C. for 15 seconds, reaction at 70° C.for 30 seconds, and reaction at 72° C. for 3 minutes; and 30 cycles eachconsisting of reaction at 94° C. for 15 seconds, reaction at 68° C. for30 seconds, and reaction at 72° C. for 3 minutes, followed by reactionat 72° C. for 10 minutes. The PCR was carried out using the PTC-200 DNAEngine (manufactured by Bio-Rad).

Next, in order to determine the nucleotide sequence by cloning, theobtained PCR product was separated by agarose gel electrophoresis, andeach of a VH gene fragment and a VL gene fragment was extracted using aGel Extraction Kit (manufactured by Qiagen). Then, deoxyadenine (dA) wasadded to the extracted fragment, using a Target Clone Plus (manufacturedby Toyobo) in accordance with the instructions attached thereto, and VHor VL of each antibody was incorporated into a pTA2 vector. AnEscherichia coli DH5α strain was transformed with the obtained vector inaccordance with the instructions attached to a Competent Quick(manufactured by Toyobo). A plasmid was extracted from the resultingtransformant using an automated plasmid isolation system (manufacturedby Kurabo). The extracted plasmid was allowed to react using a Big DyeTerminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PEBiosystems) in accordance with the instructions attached thereto andthen the nucleotide sequence of the cloned PCR product was analyzedusing a sequencer of the same company, ABI PRISM3700. As a result, aplasmid containing a full-length VH cDNA, and a plasmid containing a VLcDNA were obtained, in which an ATG sequence presumed to be aninitiation codon was present at the 5′ terminal of cDNA. A scheme ofcloning is shown in FIG. 5.

(3) Analysis of Nucleotide Sequence of Anti-CD4 Monoclonal AntibodyVariable Region

Complete nucleotide sequences of VH of the anti-CD4 rat monoclonalantibodies KM4065 to KM4069 comprised in the plasmid obtained in Example5(2) are represented by SEQ ID NOs:7 to 11, respectively, complete aminoacid sequences of VH including a signal sequence deduced from thesenucleotide sequences are represented by SEQ ID NOs:12 to 16,respectively, complete nucleotide sequences of VL are represented by SEQID NOs:17 to 21, respectively, and complete amino acid sequence of VLincluding a signal sequence deduced from these nucleotide sequences arerepresented by SEQ ID NOs:22 to 26, respectively. From the comparison ofthe obtained analysis results with the sequence data [SEQUENCES ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)] of known rat antibodies, it has become clear that each of theisolated cDNAs is a full-length cDNA encoding a variable region(hereinafter, referred to as “V region”) of anti-CD4 rat monoclonalantibodies KM4065 to KM4069 containing a secretory signal sequence; inthe H chain of KM4065, the amino acid sequence from positions 1 to 19 inthe amino acid sequence represented by SEQ ID NO:12 is the secretorysignal sequence; in the L chain of KM4065, the amino acid sequence frompositions 1 to 20 in the amino acid sequence represented by SEQ ID NO:22is the secretory signal sequence; in the H chain of KM4066, the aminoacid sequence from positions 1 to 19 in the amino acid sequencerepresented by SEQ ID NO:13 is the secretory signal sequence; in the Lchain of KM4066, the amino acid sequence from positions 1 to 19 in theamino acid sequence represented by SEQ ID NO:23 is the secretory signalsequence; in the H chain of KM4067, the amino acid sequence frompositions 1 to 18 in the amino acid sequence represented by SEQ ID NO:14is the secretory signal sequence; in the L chain of KM4067, the aminoacid sequence from positions 1 to 20 in the amino acid sequencerepresented by SEQ ID NO:24 is the secretory signal sequence; in the Hchain of KM4068, the amino acid sequence from positions 1 to 19 in theamino acid sequence represented by SEQ ID NO:15 is the secretory signalsequence; in the L chain of KM4068, the amino acid sequence frompositions 1 to 20 in the amino acid sequence represented by SEQ ID NO:25is the secretory signal sequence; in the H chain of KM4069, the aminoacid sequence from positions 1 to 19 in the amino acid sequencerepresented by SEQ ID NO:16 is the secretory signal sequence; and, inthe L chain of KM4069, the amino acid sequence from positions 1 to 20 inthe amino acid sequence represented by SEQ ID NO:26 is the secretorysignal sequence.

Then, the novelty of amino acid sequences of VH and VL of anti-CD4 ratmonoclonal antibodies KM4065 to KM4069 was investigated. Using a GCGPackage (version 9.1, manufactured by Genetics Computer Group) as asequence analysis system, the amino acid sequence database ofconventional proteins was retrieved by BLASTP method [Nucleic AcidsRes., 25, 3389 (1997)]. As a result, completely coinciding amino acidsequences were not found for both of the VH and VL, so that it wasconfirmed that the VH and VL of the anti-CD4 rat monoclonal antibodiesKM4065 to KM4069 have novel amino acid sequences.

Further, CDRs of VH and VL of respective monoclonal antibodies wereidentified by comparing them with the amino acid sequences of knownantibodies. Amino acid sequences of CDR1, CDR2 and CDR3 of VH of theanti-CD4 rat monoclonal antibody KM4065 were represented by SEQ IDNOs:27, 28 and 29, respectively, and amino acid sequences of CDR1, CDR2and CDR3 of VL thereof are represented by SEQ ID NOs:30, 31 and 32,respectively. Amino acid sequences of CDR1, CDR2 and CDR3 of VH of theanti-CD4 rat monoclonal antibody KM4066 were represented by SEQ IDNOs:33, 34 and 35, respectively, and amino acid sequences of CDR1, CDR2and CDR3 of VL thereof were represented by SEQ ID NOs:36, 37 and 38,respectively. Amino acid sequences of CDR1, CDR2 and CDR3 of VH of theanti-CD4 rat monoclonal antibody KM4067 were represented by SEQ IDNOs:39, 40 and 41, respectively, and amino acid sequences of CDR1, CDR2and CDR3 of VL thereof are represented by SEQ ID NOs:42, 43 and 44,respectively. Amino acid sequences of CDR1, CDR2 and CDR3 of VH of theanti-CD4 rat monoclonal antibody KM4068 were represented by SEQ IDNOs:45, 46 and 47, respectively, and amino acid sequences of CDR1, CDR2and CDR3 of VL thereof were represented by SEQ ID NOs:48, 49 and 50,respectively. Amino acid sequences of CDR1, CDR2 and CDR3 of VH of theanti-CD4 rat monoclonal antibody KM4069 were represented by SEQ IDNOs:51, 52 and 53, respectively, and amino acid sequences of CDR1, CDR2and CDR3 of VL thereof were represented by SEQ ID NOs:54, 55 and 56,respectively.

Example 6 Preparation of Anti-CD4 Chimeric Antibody

(1) Construction of Anti-CD4 Chimeric Antibody Expression Vector

The chimeric antibody prepared in the present invention is a chimericantibody in which a heavy chain constant region (hereinafter, referredto as “113F type”) having a high CDC activity and comprising an aminoacid sequence in which a part of an Fc region of a human IgG1 antibodyis substituted with Fc of a human IgG3 antibody disclosed inUS2007/0148165, and a light chain constant region of human κ wererespectively ligated to variable regions of heavy and light chains ofthe anti-CD4 rat monoclonal antibody obtained in Example 5(2). For thispurpose, using a high-CDC type chimeric antibody expression vectorPKTX93/113F (described in US2007/0148165) comprising a variable regionof an anti-CD20 antibody, and a pTA2 vector comprising VH or VL of eachmonoclonal antibody obtained in Example 5(2), an anti-CD4 chimericantibody expression vector was constructed in accordance with thefollowing procedure (FIGS. 5 and 6).

A reaction solution (50 μL) containing 100 ng of the TA2 vectorcontaining VH or VL of each monoclonal antibody as a template, 5 μL of10×KOD Plus buffer, 5 μL of 2 mmol/L dNTPs, 2 μL of 25 mmol/L magnesiumchloride, 2.5 μL of DMSO, 1 μL of KOD Plus polymerase (manufactured byToyobo), and 1 μL of each of 10 μmol/L primers specific to VH and VL ofeach anti-CD4 rat monoclonal antibody was prepared. Using the thusprepared solution, PCR was carried out as follows: heating at 94° C. for2 minutes, followed by 30 cycles each consisting of reaction at 94° C.for 15 seconds, reaction at 60.6° C. for 30 seconds, and reaction at 68°C. for 1 minute. Primers of VH of KM4065 are represented by SEQ IDNOs:57 and 58, and primers of VL thereof are represented by SEQ IDNOs:59 and 60; primers of VH of KM4066 are represented by SEQ ID NOs:61and 62, and primers of VL thereof are represented by SEQ ID NOs:63 and64; primers of VH of KM4067 are represented by SEQ ID NOs: 65 and 66,and primers of VL thereof are represented by SEQ ID NOs:67 and 68;primers of VH of KM4068 are represented by SEQ ID NOs:69 and 70, andprimers of VL thereof are represented by SEQ ID NOs:71 and 72; andprimers of VH of KM4069 are represented by SEQ ID NOs:73 and 74, andprimers of VL thereof are represented by SEQ ID NOs:75 and 76.

Each PCR product was purified using a PCR Purification Kit (manufacturedby Qiagen). The obtained VH of each antibody was treated withrestriction enzymes ApaI (manufactured by New England Biolabs) and NotI(manufactured by New England Biolabs), thereby obtaining a NotI-ApaIfragment of VH. In addition, VL of each antibody was treated withrestriction enzymes BsiWI (manufactured by New England Biolabs) andEcoRI (manufactured by New England Biolabs), thereby obtaining anEcoRI-BsiWI fragment of VL. Furthermore, the high-CDC type chimericantibody expression vector PKTX93/113F was also subjected to treatmentswith restriction enzymes NotI and ApaI, or EcoRI and BsiWI. As with theprocedure described in Example 1(1), a vector into which a cDNA encodingVH of anti-CD4 rat monoclonal antibodies KM4065 to KM4069 was cloned,and a vector into which a cDNA encoding VL of each of anti-CD4monoclonal rat antibodies KM4065 to KM4069 was cloned were obtained.Restriction enzymes EcoRI and NotI were added to treat the obtainedPKTX93/113F vector into which VH or VL of each antibody wasincorporated, thereby obtaining an NotI-EcoRI fragment of thePKTX93/113F vector into which an EcoRI-NotI fragment of VL and VH wereincorporated. As with the procedure described in Example 1(1), ananti-CD4 chimeric antibody expression vector was obtained into whicheach of cDNAs encoding VH and VL of each of anti-CD4 rat monoclonalantibodies KM4065 to KM4069 was cloned. A scheme for the construction ofthe vector is shown in FIG. 6. In addition, anti-CD4 chimeric antibodiesprepared from the anti-CD4 rat monoclonal antibodies KM4065 to KM4069are defined as anti-CD4 chimeric antibodies KM4045 to KM4049,respectively (Table 3).

TABLE 3 Rat monoclonal antibody Chimeric antibody KM4065 KM4045 KM4066KM4046 KM4067 KM4047 KM4068 KM4048 KM4069 KM4049

(2) Expression of Anti-CD4 Chimeric Antibody Using Animal Cells

Using the anti-CD4 chimeric antibody expression vector obtained inExample 6(1), the anti-CD4 chimeric antibody in an animal cell wasexpressed by a conventional method [Antibody Engineering, A PracticalGuide, W.H. Freeman and Company (1992)] to obtain transformants KM4045to KM4049 which produce anti-CD4 chimeric antibodies. As an animal cellfor expressing the desired antibody, a CHO/DG44 cell line in which agene of α1,6-fucosyltransferase (FUT8) was double-knockout (hereinafter,referred to as “FUT8 knockout CHO cell”) was used. It is known thatfucose is not added to a core structure of a complex typeN-glycoside-linked sugar chain of the antibody expressed in this hostcell (WO2002/31140).

(3) Obtaining of Purified Chimeric Antibody

After culturing each of the transformants KM4045 to KM4049 obtained inSection (2) by a conventional culturing method, cell suspensions wererecovered and centrifuged at 3000 rpm and 4° C. for 20 minutes torecover the culture supernatants. Then, the culture supernatants werefiltration-sterilized using a 0.22-μm pore size Millex GV filter(manufactured by Millipore). The anti-CD4 chimeric antibodies KM4045 toKM4049 were purified from the obtained culture supernatants, using aProtein A High-capacity resin (manufactured by Millipore) column inaccordance with the instructions attached thereto.

(4) Preparation of IgG1 Type Antibody and 113F Type Antibody of Anti-CD4Human Antibody 6G5

In order to compare the activity of the anti-CD4 monoclonal antibody ofthe present invention, two antibodies (hereinafter, referred to as“6G5-1” and “6G5-113F”) comprising a variable region of the conventionalanti-CD4 human antibody 6G5 and a conventional human IgG Fc region or ahigh-CDC activity 113F type Fc region were prepared.

A gene encoding VH and VL of the anti-CD4 human antibody 6G5 wasconstructed by carrying out PCR using the synthetic oligo DNAsrepresented by SEQ ID NOs:89 to 92, the synthetic oligo DNAs representedby SEQ ID NOs:83 to 88, and a T3 primer and a T7 primer positioned atboth ends of the variable region, based on the sequence informationdisclosed in US20060183195. The PCR-amplified VH or VL gene fragment ofthe 6G5 antibody was ligated into a pBluescript II sk(−) vector(manufactured by Stratagene) for subcloning. Subsequently, as with theprocedure in Example 1 or Example 6(2), a 6G5.1-1 expression vector anda 6G5.1-113F expression vector were constructed (FIG. 7). Theconstructed 6G5.1-1 expression vector was expressed in both the CHO/DG44cell and the FUT8 knockout CHO cell (WO2002/31140), and the 6G5.1-113Fexpression vector was expressed in the FUT8 knockout CHO cell.Introduction of the expression vector, obtaining of the expression cellstrain, cell culture, and purification of antibodies were carried out inthe same manner as in Example 6(2) and (3). As a result, a 6G5-1antibody (hereinafter, referred to as “6G5-1”) in which fucose is boundto N-acetylglucosamine in the reducing end in a complex typeN-glycoside-linked sugar chain of the antibody through α-1,6 bond, a6G5-1 antibody (hereinafter, referred to as “6G5-P”) to which fucose isnot bound thereto, and a 6G5-113F antibody (hereinafter, referred to as“6G5-113F”) in which fucose is not bound to a complex typeN-glycoside-linked sugar chain of the Fc region of the antibody wereobtained.

(5) Determination of Fucose Content in Anti-CD4 Chimeric Antibody

In accordance with the method described in WO2002/31140, a ratio of asugar chain in which 1-position of fucose is not bound to 6-position ofN-acetylglucosamine in the reducing end in the complex typeN-glycoside-linked sugar chain of the Fc region of each anti-CD4chimeric antibody and 6G5-1 antibody through α-bond to the entire wasexamined. The results are shown in Table 4.

From these results, it was demonstrated that fucose was not added to thechimeric antibody prepared in Example 6(3).

TABLE 4 Fucose content in anti-CD4 chimeric antibody KM4045 0% KM4046 0%KM4047 0% KM4048 0% KM4049 0% 6G5-1 100% 

Example 7 Evaluation of Activity of Anti-CD4 Chimeric Antibody

In the following sections (1) to (4), the activity evaluation wascarried out for the anti-CD4 chimeric antibodies KM4045 to KM4049obtained in Example 6 and 6G5-1, 6G5-P and 6G5-113F prepared in Example6(5).

(1) Evaluation of Binding Activity of Anti-CD4 Chimeric Antibody toRecombinant Human CD4 by Binding ELISA

The binding ELISA was carried out as in the same procedure as describedin Example 2(3). As the primary antibody, the anti-CD4 chimericantibodies KM4045 to KM4049 obtained in Example 6, and 6G5-1, 6G5-P and6G5-113F, which were serially 5-fold diluted from 1 μg/mL, were used.The results are shown in FIG. 8.

As a result, all of the anti-CD4 chimeric antibodies KM4045 to KM4049,6G5-1, 6G5-P and 6G5-113F were found to bind to the recombinant humanCD4. In addition, the anti-CD4 chimeric antibodies KM4045, KM4046,KM4048 and KM4049 were found to bind to the recombinant CD4 morestrongly than 6G5-1, 6G5-P and 6G5-113F.

(2) Biacore-Based Evaluation of Binding Activity of Anti-CD4 ChimericAntibody to Recombinant Human CD4

In order to kinetically analyze the binding activity of anti-CD4chimeric antibodies KM4045 to KM4049, 6G5-1 and 6G5-P to recombinanthuman CD4, the binding activity was measured by surface plasmonresonance method (SPR). All of the following manipulations were carriedout using a Biacore T100 (manufactured by GE Healthcare Bio-Sciences).The anti-human IgG antibody was immobilized on a CM5 sensor chip(manufactured by GE Healthcare Bio-Sciences) by an amine coupling methodusing a Human Antibody Capture Kit (manufactured by GE HealthcareBio-Sciences) in accordance with the protocols attached thereto. Anassay sample (anti-CD4 chimeric antibodies KM4045 to KM4049) was addedto be captured on the anti-human IgG antibody-immobilized chip to give100 RU (resonance unit). Thereafter, the recombinant human CD4(manufactured by R&D) diluted from 2500 ng/mL in five steps was allowedto run at a flow rate of 30 μL/min onto the chip, and the sensorgramcorresponding to each concentration was obtained. The analysis wascarried out in the 1:1 binding model using an analysis software attachedto the apparatus, Biacore T100 Evaluation software (manufactured byBiacore), thereby calculating an association rate constant ka and adissociation rate constant kd of respective antibodies for human CD4.

As a result, an association rate constant ka, a dissociation rateconstant kd, and a dissociation constant K_(D) (kd/ka) of individualantibodies thus obtained are given in Table 5.

As shown in Table 5, all of the chimeric antibodies KM4045 to KM4049exhibited a higher affinity than that of the conventional anti-CD4 humanantibody 6G5-1.

TABLE 5 Antibody ka (1/Ms) kd (1/s) K_(D) (M) KM4045 3.60E+05  2.97E−08*8.24E−14 KM4046 8.34E+05 5.12E−05 6.14E−11 KM4047 1.33E+06 4.88E−043.69E−10 KM4048 5.85E+06 3.81E−03 6.51E−10 KM4049 5.77E+06 1.16E−032.00E−10 6G5-1 5.95E+05 8.72E−04 1.46E−09 *KM4045 exhibited slowdissociation and a value which exceeds a measurable limit of adissociation rate constant kd (1 × 10⁻⁵) indicated by the maker in thissystem.

(3) Evaluation of Antibody-Dependent Cellular Cytotoxicity (ADCCActivity) of Anti-CD4 Chimeric Antibody on Human Blood Cancer Cell Lines

The ADCC activity of the anti-CD4 chimeric antibodies KM4045 to KM4049,6G5-1, 6G5-P, and 6G5-113F on the human T cell lymphoma cell lines wasmeasured in the following manner.

(3)-1 Preparation of Target Cell Suspension

Human T cell lymphoma cell lines HPB-ALL and HUT78, and T celllymphoblastic lymphoma cell line SUP-T1 were washed with PBS, washedwith a phenol red-free RPMI1640 medium (manufactured by Invitrogen,hereinafter referred to as “ADCC medium”) containing 5% dialyzed fetalbovine serum (dFBS, manufactured by Invitrogen) (hereinafter, referredto as “ADCC medium”), and then suspended in the same medium to give anoptimum concentration and used as a target cell suspension.

(3)-2 Preparation of Effector Cell Suspension

From healthy human peripheral blood, peripheral blood mononuclear cells(PBMCs) were isolated in the following manner. From healthy volunteers,50 mL of healthy human peripheral blood was collected using a syringe towhich 0.5 mL of heparin sodium (manufactured by Shimizu Pharmaceutical)was added. To the collected peripheral blood was added an equal volumeof physiological saline (manufactured by Otsuka Pharmaceutical) todilute, followed by thoroughly stirring. Then, 4.7 mL of Lymphoprep(manufactured by Axis-Shield) was dispensed into each of 15 mL tubes(manufactured by Greiner). The diluted peripheral blood (10 mL) wasgently layered on the Lymphoprep. Next, the mononuclear cell layer wasseparated by the condition of centrifugation at 2000 rpm, break off, androom temperature for 20 minutes. The thus obtained mononuclear cellfraction was washed twice with an ADCC medium, and then suspended in thesame medium to give an optimum cell count and used as an effector cellsuspension.

(3)-3 Measurement of ADCC Activity

The ADCC activity was measured by the following procedure, using aCytoTox96 Non-Radioactive Cytotoxicity Assay (manufactured by Promega)in accordance with the instructions attached thereto.

An antibody solution (50 μL/well), in which each antibody was serially20-fold diluted from 2 μg/mL or 1 μg/mL, was dispensed into a 96-well Ubottom plate (manufactured by Falcon). Next, 2.5×10⁴ cells/50 μL/well ofthe target cell suspension prepared in Section (3)-1 were dispensedthereto. Finally, 2×10⁵ cells/50 μL/well of the effector cell suspensionprepared in Section (3)-2 were dispensed thereto to give a total volumeof 150 μL, followed by reaction at 37° C. for 4 hours. Therefore, theexperiment was carried out at a 8:1 ratio of effector cell (E):targetcell (T). The ADCC activity was calculated by the following formula. Theresults are shown in FIG. 9.

ADCC activity (%)={([absorbance of sample]−[absorbance of target cellspontaneous release]−[absorbance of effector cell spontaneousrelease])/([absorbance of target cell total release]−[absorbance oftarget cell spontaneous release])}×100  (Formula)

As a result, the conventional anti-CD4 human antibody 6G5-1 exhibited noADCC activity on the human T cell lymphoma cell line HPB-ALL, whereas6G5-P and 6G5-113F, to which a core fucose was not bound to the complexN-glycoside-linked sugar chain of the Fc region of the antibody,exhibited high ADCC activity. On the other hand, the ADCC activity of6G5-P and 6G5-113F exhibited almost the same activity (FIG. 9A). Theseresults are consistent with the results disclosed in U.S. Pat. No.7,214,775 and Cancer Res 2008; 68: (10): 3863-72.

In addition, all of the anti-CD4 chimeric antibodies KM4045, KM4046,KM4047, KM4048 and KM4049 of the present invention exhibited a high ADCCactivity equal to or higher than 6G5-P, on the human T cell lymphomacell lines HUT78 and the human T cell lymphoblastic lymphoma cell lineSUP-T1 (FIGS. 9B and C). Further, all of the anti-CD4 chimericantibodies KM4045, KM4046, KM4048 and KM4049 exhibited a higher ADCCactivity than 6G5-113F, on the human T cell lymphoma cell line HPB-ALL(FIG. 9D).

From these results, it was demonstrated that all of the anti-CD4chimeric antibodies KM4045, KM4046, KM4047, KM4048 and KM4049 of thepresent invention exhibited high ADCC activity equal to or higher thanthe conventional anti-CD4 human antibody.

(4) Evaluation of Complement-Dependent Cellular Cytotoxicity (CDCActivity) of Anti-CD4 Chimeric Antibody on CD4 Transfectant and Human TCell Lymphoma Cell Line

The CDC activity of anti-CD4 chimeric antibodies KM4045 to KM4049 on theCD4 transfectant constructed in Example 1 and the human T cell lymphomacell line HPB-ALL was measured in the same manner as in Example 4(3).

As a result, the conventional anti-CD4 human antibody 6G5-1 and theconventional anti-CD4 human antibody 6G5-113F having a 113F type Fc witha partial domain-exchange of Fc of IgG1 into Fc of IgG3 exhibited no CDCactivity on the CD4 transfectant, whereas all of the anti-CD4 chimericantibodies KM4045, KM4046, KM4047, KM4048 and KM4049 of the presentinvention exhibited a CDC activity (FIG. 10A). On the other hand, allthe conventional anti-CD4 human antibody 6G5-113F having a 113F type Fcwith a partial domain-exchange of Fc of IgG1 into Fc of IgG3, theconventional anti-CD4 mouse antibody OKT4, and the anti-CD4 chimericantibodies KM4045 and KM4046 of the present invention exhibited no CDCactivity on the human T cell lymphoma cell line HPB-ALL, and only theanti-CD4 chimeric antibody KM4049 of the present invention exhibited aCDC activity on the human T cell lymphoma cell line (FIG. 10 B).

Example 8 Preparation of Humanized Antibody

(1) Design of Amino Acid Sequences of VH and VL of Anti-CD4 HumanizedAntibody

An amino acid sequence of VH of an anti-CD4 humanized antibody wasdesigned in the following manner. Firstly, in order to graft the aminoacid sequences of CDR1 to CDR3 of an anti-CD4 rat monoclonal antibodyKM4065VH represented by SEQ ID NOs:27 to 29, respectively, the aminoacid sequence of FR of VH of a human antibody was selected. Using a GCGPackage (manufactured by Genetics Computer Group) as a sequence analysissystem, a human antibody having a high homology with the anti-CD4 ratmonoclonal antibody KM4065 was retrieved by the BLASTP method [NucleicAcids Res., 25, 3389 (1997)], based on the amino acid database ofconventional proteins. When the homology score was compared with thehomology of an actual amino acid sequence, SWISSPROT database accessionno. AAC18328, Autoreactivity and immunoglobulin VH gene expression inaging humans (hereinafter, referred to as “AAC 18328”) exhibited ahomology of 84.2%. Since it was a human antibody which had the highesthomology, the amino acid sequence of FR of this antibody was selected.With regard to the FR amino acid sequence of AAC18328, it exhibited adeletion of one amino acid residue (the first residue) at the N-terminalof a secretory antibody reported by Kabat et al. [Sequences of Proteinsof Immunological Interest, US Dept. Health and Human Services (1991)].For the residue at the first position, Glu was selected which is anamino acid residue exhibited by the anti-CD4 rat monoclonal antibodyKM4065. In addition, with regard to Val at the 11^(th) position, Val wassubstituted with Leu which is an amino acid residue appearing at a highfrequency in the sequence of the human antibody and being exhibited inthe anti-CD4 rat monoclonal antibody KM4065. Since these amino acidresidues are found at a high frequency in any human antibody sequence[Sequence of Proteins of Immunological Interest, US Dept. Health andHuman Service (1990], they are not construed as being deviated from thehuman antibody sequence.

The amino acid sequences of CDRs of VH of the anti-CD4 rat monoclonalantibody KM4065 represented by SEQ ID NOs:27 to 29 were grafted into anappropriate position of the thus determined amino acid sequence of FR ofthe human antibody. In this manner, the amino acid sequence HV0 of VH ofan anti-CD4 humanized antibody represented by SEQ ID NO:77 was designed.

Next, an amino acid sequence of VL of an anti-CD4 humanized antibody wasdesigned in the following manner.

An amino acid sequence of FR of VL of a human antibody for grafting theamino acid sequences of CDR1 to CDR3 of an anti-CD4 rat monoclonalantibody KM4065VL represented by SEQ ID NOs:30 to 32, respectively, wasselected. Kabat et al. have classified VL of conventionally knownvarious human antibodies into four subgroups (HSG I to IV) based on thehomology of their amino acid sequences and reported on the consensussequences for each of the subgroups [Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)].Accordingly, the homology was examined between the amino acid sequenceof FR of consensus sequences of subgroups I to IV of VL of the humanantibody and the amino acid sequence of FR of VL of the anti-CD4 ratantibody KM4065.

As a result of retrieving the homology, the amino acid sequence of FR ofKM4065VL had a high homology of 72.2% with HSGI. Based on these results,the amino acid sequence of CDR of VL of the anti-CD4 rat monoclonalantibody KM4065 was grafted into an appropriate position of an aminoacid sequence of FR of the consensus sequence of subgroup I of VL of thehuman antibody. However, since Leu at position 24 and Leu at position128 in the amino acid sequence of VL of the anti-CD4 rat monoclonalantibody KM4065 represented by SEQ ID NO:22 are not the amino acidresidues having the highest use frequency in the region whichcorresponds to the amino acid sequence of the human antibody FR cited byKabat et al., but are amino acid residues which are used at a relativelyhigh frequency, the above-mentioned amino acid residues which arerecognized in the amino acid sequence of the anti-CD4 rat monoclonalantibody KM4065 were used. In this manner, the amino acid sequence LV0of VL of an anti-CD4 humanized antibody represented by SEQ ID NO:78 wasdesigned.

The amino acid sequence HV0 of VH and the amino acid sequence LV0 of VLof the anti-CD4 humanized antibody designed in the above are sequencesin which the CDR amino acid sequence of the anti-CD4 rat monoclonalantibody KM4065 alone was grafted into the selected human antibody FRamino acid sequence, but in general, when a humanized antibody isprepared, its binding activity is frequently lowered in the case ofmerely a simple grafting of a CDR amino acid sequence of a mouseantibody to a human antibody FR. For these reasons, in order to avoiddecreasing of the binding activity, modifications of the amino acidresidues considered to have an influence on the binding activity, amongthe FR amino acid residues which are different between human antibodiesand mouse antibodies, are carried out together with the grafting of theCDR amino acid sequences. Thus, also in this example, the amino acidresidues of FR considered to have an influence on the binding activitywere identified in the following manner.

Firstly, a three-dimensional structure of an antibody V region (HV0LV0)consisting of the amino acid sequence HV0 of VH and the amino acidsequence LV0 of VL of the anti-CD4 humanized antibody designed in theabove was constructed using a computer modeling technique. Preparationof the three dimensional structure coordinates was carried out using asoftware AbM (manufactured by Oxford Molecular) in accordance withinstructions attached thereto, and display of the three-dimensionalstructure was carried out using a software Pro-Explore (manufactured byOxford Molecular) or ViewerLite (manufactured by Accelrys) in accordancewith instructions attached thereto. In addition, a computer model of thethree-dimensional structure of the V region of the anti-CD4 ratmonoclonal antibody KM4065 was also constructed in the same manner.Further, by similarly constructing a three-dimensional structure modelconsisting of an amino acid sequence in which the amino acid residues inthe FR amino acid sequences of VH and VL of HV0LV0, which are differentfrom those of the anti-CD4 rat antibody KM4065, were selected andmodified into the amino acid residues of the anti-CD4 rat monoclonalantibody KM4065, three-dimensional structures of the V regions of theanti-CD4 rat monoclonal antibody KM4065, HV0LV0 and modified productwere compared, whereby the amino acid residues predicted to have aninfluence on the binding activity of the antibody were identified.

As a result, as the amino acid residues among amino acid residues of FRof HV0LV0, which are considered to change a three-dimensional structureof the antigen binding region and therefore have an influence on thebinding activity of the antibody, Leu at position 18, Val at position93, Ala at position 97, and Thr at position 114 in the HV0 sequence, andAla at position 13, Val at position 15, Val at position 19, Ala atposition 47, Val at position 62, and Leu at position 82 in the LV0sequence were respectively selected. By modifying at least one or moreamino acid sequences of these selected amino acid residues into theamino acid residues which are present at the same positions of the aminoacid sequence of the anti-CD4 rat monoclonal antibody KM4065, VH and VLof a humanized antibody having various modifications were designed.Specifically, regarding the antibody VH, at least one modification wasintroduced among the amino acid modifications for substituting Leu atposition 18 with Met, Val at position 93 with Thr, Ala at position 97with Thr, and Thr at position 114 with Ile in the amino acid sequencerepresented by SEQ ID NO:77. Further, regarding the antibody VL, atleast one modification was introduced among the amino acid modificationsfor substituting Ala at position 13 with Val, Val at position 15 withLeu, Val at position 19 with Ala, Ala at position 47 with Gln, Val atposition 62 with Be, and Leu at position 82 with Val in the amino acidsequence represented by SEQ ID NO:78.

By designing amino acid sequences of variable regions of HV2LV0, HV3LV0,HV4LV0, and HV4LV6 with modifications of at least one of amino acidresidues present in FR of HV0LV0, amino acid sequences of H chainvariable regions HV2, HV3 and HV4, and L chain variable region LV6 arerepresented by SEQ ID NOs:96, 98, 100 and 102, respectively.

(2) Preparation and Evaluation of Anti-CD4 Humanized Antibody

A DNA encoding the amino acid sequence of the variable region of theanti-CD4 humanized antibody was constructed in mammalian cells using acodon which is used at a high frequency, when amino acid modification(s)are carried out using a codon which is used as a DNA encoding the aminoacid sequence of VH or VL of the anti-CD4 rat monoclonal antibodyKM4065. DNA sequences encoding the amino acid sequence of HV0 and LV0 ofthe anti-CD4 humanized antibody were represented by SEQ ID NOs:93 and94, respectively, whereas DNA sequences encoding the amino acidsequences of variable regions HV2, HV3, HV4, and LV6 in which amino acidmodification(s) were introduced were represented by SEQ ID NOs:95, 97,99 and 101, respectively. Each DNA sequence was constructed by thecomplete synthesis. As in the same procedure described in Example 6, thecomplete synthesis DNA encoding each variable region was inserted intothe humanized antibody expression vector pKANTEX93 to construct ananti-CD4 humanized antibody expression vector. The constructed anti-CD4humanized antibody expression vector was introduced into an animal cellto express a humanized antibody, thereby preparing anti-CD4 humanizedantibodies HV0LV0, HV2LV0, HV3LV0, HV4LV0 and HV4LV6.

When the binding activity of the obtained anti-CD4 humanized antibodieswas measured using a Biacore T100, all of the prepared anti-CD4humanized antibodies HV2LV0, HV3LV0, HV4LV0 and HV4LV6 exhibited abinding activity equal to or ½ or higher than that of the anti-CD4chimeric antibody KM4045. Therefore, an anti-CD4 humanized antibodyhaving a high binding activity was prepared.

Example 9 Evaluation of Binding Activity of Humanized Antibody

The binding activity evaluation was carried out for the anti-CD4humanized antibodies HV0LV0, HV2LV0, HV3LV0, HV4LV0 and HV4LV6 preparedaccording to the method described in Example 8.

(1) Biacore-Based Evaluation of Binding Activity of Anti-CD4 HumanizedAntibody to Recombinant Human CD4

The measurement of a binding activity was carried out according to themethod described in Example 7(2). The association rate constant ka, thedissociation rate constant kd and the dissociation constant K_(D)(kd/ka) of respective antibodies thus obtained are shown in Table 6.

As a result, the humanized antibodies HV0LV0, HV2LV0, HV3LV0, HV4LV0,and HV4LV6 maintained a very strong binding activity of 1×10⁻¹⁰M orhigher. This is a binding activity equivalent to that of the chimericantibody KM4045. And the binding activity is higher than that of 6G5-1.

TABLE 6 Antibody ka (1/Ms) kd (1/s) K_(D) (M) KM4045 5.41E+05 <1E−05*<1.84E−11* HV0LV0 1.47E+06 2.86E−05   1.95E−11 HV2LV0 1.14E+06 <1E−05*<8.77E−12* HV3LV0 1.33E+06 <1E−05* <7.52E−12* HV4LV0 7.91E+05 <1E−05*<1.26E−11* LV4LV6 4.03E+05 <1E−05* <2.48E−11* 6G5-1 5.34E+05 8.24E−04  1.54E−09 *Since KM4045, HV2LV0, HV3LV0, HV4LV0, and HV4LV6 exhibitedslow dissociation, the value exceeds a measurable limit of adissociation rate constant kd (1 × 10⁻⁵) indicated by the maker in thissystem.

(2) Evaluation of Binding Activity to CD4-Positive T Cell Lymphoma CellLine Using Flow Cytometer

Reactivity of various anti-CD4 antibodies to cells was evaluated.Chimeric antibody KM4045, humanized antibodies HV0LV0, HV2LV0, HV3LV0,HV4LV0 and HV4LV6, and human antibodies 6G5-1 and 6G5-P were used as theantibodies for evaluation.

In order to inhibit non-specific binding of a test antibody, a human IgG(manufactured by Sigma) at a final concentration of 1 mg/mL was added to1 to 5×10⁵ cells of the human T cell lymphoma cell line HPB-ALL. Inaddition, various anti-CD4 antibodies were appropriately diluted withBSA-PBS and added thereto to give a total volume of 50 μL. The resultingcell suspension was reacted on ice for 90 minutes, and washed two timeswith BSA-PBS. To the cells, 50 μL of an FITC-labeled anti-human IgG Fcantibody (manufactured by Acris) diluted with BSA-PBS was added,followed by reaction on ice for 90 minutes. The cells were washed oncewith BSA-PBS and then suspended in BSA-PBS. The fluorescence intensitywas measured by a flow cytometer (manufactured by Beckman Coulter). FIG.11 shows the mean fluorescence intensity (MFI value) when the antibodywas reacted at a concentration of 6.7, 0.67 and 0.067 μg/mL. All of theanti-CD4 humanized antibodies bound to the CD4-positive cell line in anantibody concentration-dependent manner. Each of the binding degrees wasequivalent to that of the chimeric antibody KM4045.

Example 10 Evaluation of In Vitro Medicinal Efficacy of HumanizedAntibody HV2LV0

The anti-CD4 humanized antibody HV2LV0 prepared in Example 8 ishereinafter referred to as KM8045. The antibody-dependent cellularcytotoxicity (ADCC activity) of the anti-CD4 humanized antibody on ahuman blood cancer cell line was evaluated according to the methoddescribed in Example 7(3). The target cell employed three types ofcells, i.e., human T cell lymphoma cell lines HPB-ALL, HUT78, andCCRF-CEM. In addition, the antibody employed the humanized antibodyKM8045, the chimeric antibody KM4045, and the human antibodies 6G5-1 and6G5-P.

As a result, the humanized antibody KM8045 exhibited on any cell line, ahigh ADCC activity equivalent to that of the chimeric antibody KM4045,and also exhibited an ADCC activity equal to or higher than that of6G5-P. 6G5-1 exhibited only a weak ADCC activity on HPB-ALL and HUT78,and exhibited no ADCC activity on CCRF-CEM (FIG. 12).

Example 11 Evaluation of In Vivo Medicinal Efficacy of HumanizedAntibody KM8045

(1) Mouse Early Stage Cancer Model Using Human T Cell Lymphoma Cell LineHH as target The in vitro-cultured human T cell lymphoma cell line HHwas suspended at a density of 1×10⁸ cells/mL in RPMI1640 medium (GibcoBRL). Into the right abdominal of SCID mice (male, 8 weeks old,purchased from CLEA Japan), 100 μL (1×10⁷ cells/mouse) of the suspensionwas subcutaneously transplanted. The anti-CD4 humanized antibody KM8045or anti-CD4 human antibody 6G5-P was diluted to give 1 μg/100 μL/headand intravenously administered to the mice on Days 0, 3, and 5, threetimes in total. A negative control group was given PBS alone. The day oftumor transplantation was taken as Day 0, and the diameter of tumor masswas measured day by day using vernier calipers. The tumor volume wascalculated according to the following formula.

tumor volume=breadth×breadth×length×0.5

Changes over days in a mean value of the tumor volume of each group areshown in FIG. 13. As shown in FIG. 13, the 6G5-P-administered groupexhibited only weak anti-tumor effects as compared to the negativecontrol group, whereas the KM8045-administered group exhibitedsignificant anti-tumor effects.

From these results, it was demonstrated that the anti-CD4 humanizedantibody KM8045 has higher anti-tumor effects than 6G5-P, on the mouseearly stage cancer model in which an HH cell is used as a target.

(2) Mouse Metastasis Model Using CD4-Expressing EL4 as Target

CD4/EL4 constructed in Example 1(3) was cultured in vitro and suspendedat a density of 1×10⁶ cells/mL in RPMI1640 medium (Gibco BRL). Into thetail vein of C57BL/6 mice (male, 8 weeks old, purchased from CLEA Japan)was transplanted 100 μL/head (1×10⁵ cells/head) of the cell suspension.The day of tumor transplantation was defined as Day 0, and on Day 1.Each of anti-CD4 humanized antibody KM8045, and anti-CD4 humanantibodies 6G5-P and 6G5-1 was prepared to be 0.2 μg/100 μL/head and 2μg/100 μL/head, and then intravenously administered at a dose of 100μL/head to the mice. The negative control group was given PBS alone, oran isotype control antibody. On Day 14, a body weight of all the micewas measured, and the mice were sacrificed by bleeding under etheranesthesia, followed by cervical dislocation. Thereafter, the liver andthe kidney were collected and weighed. Then, a ratio of the liver weightand the kidney weight relative to the body weight of individual mice(hereinafter, referred to as “liver weight ratio” and “kidney weightratio”) was calculated as a percentage. The evaluation of anti-tumoreffects was carried out by comparing the liver and kidney weight ratioof each group which was increased due to the metastasis of tumor cells,with the liver and kidney weight ratio measured at the same time (meanvalue of three mice) of healthy mice which tumor had not beentransplanted. The liver weight ratio and the kidney weight ratio of eachgroup are shown in FIGS. 14A and 14B, respectively. As shown in FIGS.14A and 14B, the 6G5-1-administered group exhibited no anti-tumoreffects relative to the negative control group, whereas the6G5-P-administered group and the KM8045-administered group exhibitedanti-tumor effects. Further, more potent anti-tumor effects wererecognized in the KM8045-administered group than in the6G5-P-administered group.

From these results, it was demonstrated that KM8045 has higheranti-tumor effects than 6G5-1 and 6G5-P, on the mouse metastasis modelwhen CD4/EL4 is used as a target.

(3) Mouse Progressive Cancer Model Using Human T Cell Lymphoma Cell LineHH as Target

As with Example 11(3), the in vitro-cultured HH cell line was suspendedat a density of 1×10⁸ cells/mL in RPMI1640 medium (Gibco BRL). Into theright abdominal of SCID mice (male, 8 weeks old, purchased from CLEAJapan), 100 μL/head (1×10⁷ cells/head) of the suspension wassubcutaneously transplanted. On Day 6 at which a tumor volume became anaverage of 60 mm³, mice were grouped. The anti-CD4 humanized antibodyKM8045 was diluted with PBS to give a dose of 20, 100, and 200 μg/100μL/head, and intravenously administered to the mice twice a week, sixtimes in total. The negative control group was administered PBS alone.The day of tumor transplantation was defined as Day 0, and the diameterof tumor mass was measured day by day using vernier calipers. The tumorvolume was calculated according to the following formula.

Tumor Volume=Breadth×Breadth×Length×0.5

Changes over days in a mean value of the tumor volume of each group areshown in FIG. 15. As shown in FIG. 15, the KM8045-administered groupexhibited anti-tumor effects. From the observation, that anti-tumoreffect in the 200 μg/head group is higher than that in 20 μg/head and100 μg/head groups, dose-dependent anti-tumor effects were confirmed.

From these results, it was demonstrated that the anti-CD4 humanizedantibody KM8045 also had anti-tumor effects on the progressive cancermodel in which an HH cell was used as a target.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese application No. 2008-331904, filedon Dec. 26, 2008, and U.S. provisional application No. 61/141,393, filedon Dec. 30, 2008, the entire contents of which are incorporated hereintoby reference. All references cited herein are incorporated in theirentirety.

1-20. (canceled)
 21. A DNA encoding a monoclonal antibody or antibodyfragment thereof, wherein said monoclonal antibody or antibody fragmentthereof comprises a heavy chain variable region (VH) and a light chainvariable region (VL), and wherein said monoclonal antibody or antibodyfragment thereof is selected from the group consisting of (a)-(d): (a) amonoclonal antibody or antibody fragment thereof in which the VH and VLcomprise the amino acid sequences of SEQ ID NOs: 16 and 26,respectively, or the VH and VL comprise the amino acid sequences of SEQID NOs: 12 and 22, respectively; (b) a monoclonal antibody or antibodyfragment thereof in which CDRs 1 to 3 of the VH comprise the amino acidsequences of SEQ ID NOs: 51 to 53, respectively, and CDRs 1 to 3 of theVL comprise the amino acid sequences of SEQ ID NOs: 54 to 56,respectively; (c) a monoclonal antibody or antibody fragment thereof inwhich CDRs 1 to 3 of the VH comprise the amino acid sequences of SEQ IDNOs: 27 to 29, respectively, and CDRs 1 to 3 of the VL comprise theamino acid sequences of SEQ ID NOs: 30 to 32, respectively; and (d) amonoclonal antibody or antibody fragment thereof selected from the groupconsisting of (1)-(5): (1) a humanized antibody in which the VH and VLcomprise the amino acid sequences of SEQ ID NOs: 77 and 78,respectively; (2) a humanized antibody in which the VH and VL comprisethe amino acid sequences of SEQ ID NOs: 96 and 78, respectively; (3) ahumanized antibody in which the VH and VL comprise the amino acidsequences of SEQ ID NOs: 98 and 78, respectively; (4) a humanizedantibody in which the VH and VL comprise the amino acid sequences of SEQID NOs: 100 and 78, respectively; and (5) a humanized antibody in whichthe VH and VL comprise the amino acid sequences of SEQ ID NOs: 100 and102, respectively.
 22. A recombinant vector which comprises the DNAaccording to claim
 21. 23. A transformant obtainable by introducing therecombinant vector according to claim 22 into a host cell.
 24. A processfor producing a monoclonal antibody or antibody fragment thereof thatbinds to an extracellular region of CD4, said process comprisingculturing the transformant according to claim 23 in a culture medium toproduce and accumulate the monoclonal antibody or antibody fragmentthereof, and collecting the antibody or antibody fragment thereof fromthe culture medium.